U.S. patent application number 11/014842 was filed with the patent office on 2006-01-19 for recombinant measles viruses expressing epitopes of antigens of rna viruses - use for the preparation of vaccine compositions.
Invention is credited to Frederic Delebecque, Clarisse Lorin, Lucile Mollet, Frederic Tangy.
Application Number | 20060013826 11/014842 |
Document ID | / |
Family ID | 29716945 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060013826 |
Kind Code |
A1 |
Tangy; Frederic ; et
al. |
January 19, 2006 |
Recombinant measles viruses expressing epitopes of antigens of RNA
viruses - use for the preparation of vaccine compositions
Abstract
The invention relates to a recombinant measles virus expressing
a heterologous amino acid sequence derived from an antigen of a
determined RNA virus, said recombinant measles virus being capable
of eliciting a humoral and/or cellular immune response against
measles virus or against said RNA virus or against both measles
virus and against said RNA virus. It also relates to the use of
said recombinant measles virus for the preparation of immunogenic
composition.
Inventors: |
Tangy; Frederic; (Les Lilas,
FR) ; Lorin; Clarisse; (Paris, FR) ; Mollet;
Lucile; (Paris, FR) ; Delebecque; Frederic;
(Paris, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
29716945 |
Appl. No.: |
11/014842 |
Filed: |
December 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/EP03/07146 |
Jun 20, 2003 |
|
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11014842 |
Dec 20, 2004 |
|
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Current U.S.
Class: |
424/199.1 ;
435/235.1; 435/5 |
Current CPC
Class: |
A61K 47/6901 20170801;
C12N 2760/18443 20130101; C12N 15/86 20130101; C12N 2760/18434
20130101; C12N 2740/16022 20130101; C12N 2760/18421 20130101; C12N
2770/24122 20130101; A61K 39/12 20130101; A61P 31/12 20180101; C12N
2740/16122 20130101; A61P 37/04 20180101; C07K 14/005 20130101;
A61K 39/165 20130101; A61K 39/21 20130101; C12N 7/00 20130101; C12N
2760/18422 20130101; A61K 2039/5256 20130101; C12N 2760/18452
20130101; A61P 31/14 20180101; Y02A 50/30 20180101 |
Class at
Publication: |
424/199.1 ;
435/005; 435/235.1 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; A61K 39/12 20060101 A61K039/12; C12N 7/00 20060101
C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2002 |
EP |
02291550.8 |
Claims
1. Recombinant mononegavirales virus expressing a heterologous
amino acid sequence, said recombinant virus being capable of
eliciting a humoral and/or a cellular immune response against said
heterologous amino acid sequence including in individuals having
pre-existing measles virus immunity.
2. Recombinant measles virus expressing a heterologous amino acid
sequence derived from an antigen of a determined RNA virus, said
recombinant measles virus being capable of eliciting a humoral
and/or a cellular immune response against measles virus or against
said RNA virus or against both measles virus and against said RNA
virus.
3. Recombinant measles virus according to claim 2 which is the
product of the expression in a cell of a recombinant nucleotide
sequence comprising acDNA molecule encoding the full length
antigenomic (+) RNA of the measles virus (MV) and further
comprising, recombined with said cDNA molecule, a sequence encoding
an heterologous amino acid sequence of a determined retrovirus or
flavivirus.
4. Recombinant measles virus according to claims 1 or 2, which is
derived from the Schwarz vaccine strain or from the Edmonston
strain.
5. Recombinant measles virus according to anyone of claims 2 to 4,
which is rescued from helper cells transfected with a recombinant
nucleotide sequence which comprises cDNA encoding the nucleotid
sequence of full length antigenomic (+) RNA of the measles virus,
said cDNA being recombined with a nucleotide sequence encoding a
retroviral orflaviviral heterologous amino acid sequence, and said
recombinant nucleotide sequence complying with the rule of six.
6. Recombinant measles virus according to anyone of claims 2 to 5,
wherein the nucleotide sequence is comprised within the EdB-tag
virus vector recombined with the ATU inserted in a position of the
EdB-tag vector taking advantage of the gradient of the viral genome
to allow various levels of expression of the transgenic nucleotide
sequence encoding the heterologous amino acid sequence inserted in
said ATU.
7. Recombinant measles virus according to anyone of claims 2 to 5,
which is the product of the expression of a recombinant nucleotide
sequence comprising a cDNA molecule which encodes the nucleotide
sequence of the full length antigenomic (+) RNA of a measles virus
(MV) originating from an approved vaccine strain, wherein said cDNA
molecule is recombined with a heterologous nucleotide sequence
encoding a heterologous amino acid sequence derived from an antigen
of a determined heterologous retrovirus or flavivirus.
8. Recombinant measles virus according to claim 7, wherein the cDNA
molecule comprises the insert contained in plasmid pTM-MVSchw
deposited on Jun. 12, 2002 under No. 1-2889, wherein said insert
encodes the nucleotide sequence of the full length antigenomic (+)
RNA strand of the measles virus.
9. Recombinant measles virus according to claim 7 or 8, wherein the
recombinant nucleotide sequence is derived from the insert
contained in plasmid pTM-MVSchw2-gfp deposited on Jun. 12, 2002
under I-2890 (CNCM), wherein the sequence of the gfp gene is
substituted for a sequence encoding a determined amino acid
sequence.
10. Recombinant measles virus according to anyone of claims 7 to 9,
wherein the cDNA molecule is selected among the following
sequences: nucleotide sequence extending from nucleotide 83 to
nucleotide 15977 of FIG. 11 nucleotide sequence extending from
nucleotide 29 to nucleotide 15977 of FIG. 11 nucleotide sequence
extending from nucleotide 29 to nucleotide 16202 of FIG. 11
nucleotide sequence extending from nucleotide 26 to nucleotide
15977 of FIG. 11 nucleotide sequence extending from nucleotide 26
to nucleotide 16202 of FIG. 11 nucleotide sequence extending from
nucleotide 9 to nucleotide 15977 of FIG. 11 nucleotide sequence
extending from nucleotide 9 to nucleotide 16202 of FIG. 11
11. Recombinant measles virus according to anyone of claims 2 to
10, wherein the heterologous amino acid sequence is derived from an
antigen of a retrovirus selected among HIV retroviruses, or from an
antigen of a flavivirus.
12. Recombinant measles virus according to anyone of claims 2 to 9,
wherein the heterologous amino acid sequence is derived from an
antigen of the Yellow Fever Virus or the West Nile Virus.
13. Recombinant measles virus according to anyone of claims 2 to
11, wherein the heterologous amino acid sequence is derived from an
envelope antigen of the HIV retrovirus.
14. Recombinant measles virus according to claim 13, wherein the
heterologous amino acid sequence is a recombinant gp160 or a
recombinant gp120 antigens of HIV-1.
15. Recombinant measles virus according to claim 14, wherein the
V1, V2 and/or V3 loops of the gp120 antigen are deleted or deleted
in part, individually or in combination in such a way that
conserved epitopes are exposed on the obtained recombinant gp120
antigen.
16. Recombinant measles virus according to claim 15, wherein the
V1, V2 and/or V3 loops of the gp120 antigen are substituted or
substituted in part, individually or in combination in such a way
that conserved epitopes are exposed on the obtained recombinant
gp120 antigen.
17. Recombinant measles virus according to claim 16, wherein the V3
loop is substituted for a gp41 epitope such as AAELDKWASAA (SEQ ID
NO: 8).
18. Recombinant measles virus according to anyone of claims 11 to
16, wherein the heterologous amino acid sequence isgp160 .DELTA.V3,
gp160 .DELTA.V1V2, gp160 .DELTA.V1V2V3, gp140 .DELTA.V3, gp140
.DELTA.V1V2, gp140 .DELTA.V1V2V3.
19. Recombinant measles virus according to anyone of claims 2 to
12, wherein the amino acid sequence is derived from an antigen of
the Yellow Fever virus selected among the envelope (Env) or the NS1
proteins or immunogenic mutants thereof.
20. Recombinant measles virus according to anyone of claims 2 to
12, wherein the amino acid sequence is derived from an antigen of
the West Nile virus selected among the envelope (E),
premembrane(preM) or immunogenic mutants thereof.
21. Recombinant measles virus according to anyone of claims 2 to
12, capable of inducing protection against heterologous VLP or pM/E
proteins.
22. Recombinant measles virus according to anyone of claims 2 to
21, which elicits a humoral and/or a cellular immune response in an
animal model susceptible to measles virus.
23. Recombinant measles virus according to anyone of claims 2 to
22, which elicits neutralizing antibodies against the heterologous
amino acid sequence in a mammalian animal model susceptible to
measles virus.
24. Recombinant measles virus according to anyone of claims 2 to 23
wherein the heterologous amino acid sequence is derived from an
envelope proteins of HIV-1 and which elicits antibodies capable of
neutralizing a primary HIV isolate when tested on indicator cells
such as P4-CCR5 cells.
25. Recombinant measles virus according to claims 2 to 24 which
elicits neutralizing antibodies against the heterologous amino acid
sequence in a mammal.
26. Recombinant measles virus vector comprising a replicon
comprising (i) a cDNA sequence encoding the full length antigenomic
(+) RNA of a measles virus operatively linked to (ii) expression
control sequences and (iii) a heterologous DNA sequence coding for
a determined heterologous amino acid sequence, said heterologous
DNA sequence being cloned in said replicon in conditions allowing
its expression and said replicon having a total number of
nucleotides which complies with the rule of six.
27. Recombinant measles virus vector according to claim 26, wherein
the heterologous DNA sequence is cloned within an Additional
Transcription Unit (ATU) inserted in the cDNA corresponding to the
antigenomic RNA of measles virus.
28. Recombinant measles virus vector according to claim 26, wherein
the cloning site of the ATU is chosen upstream from the N gene of
the MV virus.
29. Recombinant measles virus vector according to claim 26, wherein
the cloning site of the ATU is chosen between the P and M genes of
the MV virus.
30. Recombinant measles virus vector according to claim 26, wherein
the cloning site of the ATU is between the H and L genes of the MV
virus.
31. Recombinant measles virus vector according to anyone of claims
26 to 29, wherein the heterologous DNA sequence is expressed as a
fusion protein with one of the MV proteins.
32. Recombinant measles virus vector according to anyone of claims
26 to 29, wherein the heterologous DNA sequence is not expressed as
a fusion protein with one of the MV proteins.
33. Recombinant measles virus vector according to anyone of claims
26 to 29 wherein the heterologous DNA sequence encodes a retroviral
amino acid sequence.
34. Recombinant measles virus vector according to anyone of claims
26 to 32 wherein the heterologous DNA sequence encodes a retroviral
amino acid sequence derived from an antigen of a retrovirus
selected among HIV retroviruses.
35. Recombinant measles virus vector according to anyone of claims
26 to 32 wherein the heterologous DNA sequence encodes a retroviral
amino acid sequence derived from an antigen of a retrovirus
selected among flaviviruses.
36. Recombinant measles virus vector according to anyone of claims
26 to 33 wherein the heterologous DNA sequence encodes a retroviral
amino acid sequence derived from an envelope antigen of an HIV
retrovirus.
37. Recombinant measles virus vector according to anyone of claims
26 to 33 wherein the heterologous DNA sequence encodes a retroviral
amino acid sequence selected among the gp160, the gp120 orgp41 of
HIV-1, or the gp140 of HIV-1, or a mutated version of said
antigens.
38. Recombinant measles virus vector according to anyone of claims
26 to 36, wherein the mutated antigen enables exposition of
neutralizing epitopes.
39. Recombinant measles virus vector according to anyone of claims
26 to 37, wherein the heterologous DNA sequence encodes gp160AV3,
gp160 .DELTA.V1V2, gp160 .DELTA.V1V2V3, gp140 .DELTA.V3, gp140
.DELTA.V1V2, gp140 .DELTA.V1V2V3.
40. Recombinant measles virus vector according to anyone of claims
25 to 32 wherein the heterologous DNA sequence encodes a flaviviral
amino acid sequence derived from an antigen of the Yellow Fever
virus or the West Nile virus.
41. Recombinant measles virus vector according to anyone of claims
26 to 40 wherein the nucleotide sequence comprising cDNA resulting
from reverse transcription of the antigenic RNA of measles virus,
originates from a measles virus strain approved for
vaccination.
42. Recombinant measles virus vector according to claim 41, wherein
the measles virus strain is the Schwarz strain.
43. Recombinant measles virus vector according to claim 42, wherein
the cDNA encoding the full length antigenomic (+) RNA of the
measles virus and the expression control sequence are derived from
pTM-MVSchw deposited at the CNCM under No. 1-2889.
44. Recombinant measles virus vector according to anyone of claims
26 to 42, which is a plasmid.
45. Recombinant measles virus vector according to anyone of claims
26 to 44 wherein the replicon is designed according to the map of
FIG. 2 wherein insert represents the heterologous DNA sequence.
46. Recombinant measles virus vector according to anyone of claims
26 to 45, wherein the heterologous DNA sequence is selected among
YFV17D204.
47. Recombinant measles virus vector according to anyone of claims
26 to 45, wherein the heterologous DNA sequence is selected among
the neurovirulent strain IS-98-ST1.
48. Recombinant measles virus vector according to claim 43, which
is selected among the following vectors deposited with the CNCM
TABLE-US-00020 pMV2 (EdB)gp160 [delta] V3HIV89. 6P CNCM 1-2883 pMV2
(EdB) gp160HIV89. 6P CNCM 1-2884 pMV2 (EdB) gp140HIV89. 6P CNCM
1-2885 pMV3 (EdB)gp140 [delta] V3HIV89. 6P CNCM 1-2886
pMV2(EdB)-NS1YFV17D CNCM I-2887 pMV2 (EdB)-EnvYFV17D CNCM 1-2888.
pTM-MVSchw2-Es(WNV) CNCM I-3033 pTM-MVSchw2-GFPbis- CNCM 1-3034
pTM-MVSchw2-p17p24[delta] myr(HIVB) CNCM 1-3035
pTM-MVSchw3-Tat(HIV89-6p) CNCM I-3036 pTM-MVschw3-GFP CNCM 1-3037
pTM-MVSchw2-Es (YFV) CNCM 1-3038 pTM-MVSchw2-gp140 [delta] V1 V2
V3(HIV89-6) CNCM 1-3054 pTM-MVSchw2-gp140 [delta] V3(HIV89-6) CNMC
I-3055 pTM-MVSchw2-gp160 [dela] V1 V2 V3(HIV89-6) CNCM 1-3056
pTM-MVSchw2-gp160 [delta] V1 V2(HIV89-6) CNCM 1-3057
pTM-MVSchw2-Gag SIV239 p17-p24 [delta] myr-3-gp CNCM I-3058. 140
(HIV89-6)
49. A rescue system for the assembly of recombinant measles virus
expressing a heterologous amino acid sequence, which comprises a
determined cell transfected with a recombinant measles virus vector
according to anyone of claims 26 to 47, and a determined helper
cell recombined with at least one vector suitable for expression of
T7 RNA polymerase and expression of the N, P and L proteins of the
measles virus.
50. An immunogenic composition comprising a recombinant virus
according to anyone of claims 1 to 26, or a recombinant vector
according to anyone of claims to 48.
51. A vaccine composition comprising a recombinant virus according
to anyone of claims 1 to 26, or a recombinant vector according to
anyone of claims to 48.
Description
[0001] The invention relates to recombinant measles viruses
expressing epitopes of antigens of RNA viruses including especially
retroviruses and flaviviruses and to their use for the preparation
of vaccine compositions.
[0002] Measles virus is a member of the order mononegavirales,
i.e., viruses with a non-segmented negative-strand RNA genome. The
non segmented genome of measles virus (MV) has an antimessage
polarity which results in a genomic RNA which is not translated
either in vivo or in vitro nor infectious when purified.
[0003] Transcription and replication of non-segmented (-) strand
RNA viruses and their assembly as virus particles have been studied
and reported especially in Fields virology (3.sup.rd edition, vol.
1, 1996, Lippincott--Raven publishers--Fields B N et al.,
Transcription and replication of measles virus do not involve the
nucleus of the infected cells but rather take place in the
cytoplasm of said infected cells. The genome of the measles virus
comprises genes encoding six major structural proteins from the six
genes (designated N, P, M, F, H and L) and an additional two-non
structural proteins from the P gene. The gene order is the
following: 3'-I, N, P (including C and V), M, F, H, and L large
polymerase protein at the 5' end. The genome further comprises non
coding regions in the intergenic region M/F; this non-coding region
contains approximately 1000 nucleotides of untranslated RNA. The
cited genes respectively encode the leader peptide (I gene), the
proteins of the nucleocapsid of the virus, i.e., the nucleoprotein
(N), the phosphoprotein (P), and the large protein (L) which
assemble around the genome RNA to provide the nucleocapsid. The
other genes encode the proteins of the viral envelope including the
hemagglutinin (H), the fusion (F) and the matrix (M) proteins.
[0004] The measles virus has been isolated and live attenuated
vaccines have been derived from the Edmonston MV isolated in 1954
(Enders, J. F., and T. C. Peebles. 1954. Propagation in tissue
cultures od cytopathogenic agents from patients with measles. Proc.
Soc. Exp. Biol. Med. 86:277-286.), by serial passages on primary
human kidney or amnion cells. The used strains were then adapted to
chick embryo fibroblasts (CEF) to produce Edmonston A and B seeds
(Griffin, D., and W Bellini. 1996. Measles virus, p. 1267-1312. In
B. Fields, D. Knipe, et al. (ed.), Virology, vol. 2.
Lippincott--Raven Publishers, Philadelphia). Edmonston B was
licensed in 1963 as the first MV vaccine. Further passages of
Edmonston A and B on CEF produced the more attenuated Schwarz and
Moraten viruses (Griffin, D., and W. Bellini. 1996. Measles virus,
p. 1267-1312. In B. Fields, D. Knipe, et al. (ed.), Virology, vol.
2. Lippincott--Raven Publishers, Philadelphia) whose sequences have
recently been shown to be identical (Parks, C. L., R. A. Lerch, P.
Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Analysis of
the noncoding regions of measles virus strains in the Edmonston
vaccine lineage. J. Virol. 75:921-933; Parks, C. L., R. A. Lerch,
P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001.
Comparison of predicted amino acid sequences of measles virus
strains in the Edmonston vaccine lineage. J. Virol. 75:910-920).
Because Edmonston B vaccine was reactogenic, it was abandoned in
1975 and replaced by the Schwarz/Moraten vaccine which is currently
the most widely used measles vaccine in the world (Hilleman, M.
2002. Current overview of the pathogenesis and prophylaxis of
measles with focus on practical implications. Vaccine. 20:651-665).
Several other vaccine strains are also used: AIK-C, Schwarz F88,
CAM70, TD97 in Japan, Leningrad-16 in Russia, and Edmonston Zagreb.
The CAM70 and TD97 Chinese strains were not derived from Edmonston.
Schwarz/Moraten and AIK-C vaccines are produced on CEF. Zagreg
vaccine is produced on human diploid cells (WI-38).
[0005] The live attenuated vaccine derived from the Schwarz strain
is commercialized by Aventis Pasteur (Lyon France) under the
trademark Rouvax.RTM..
[0006] In a noteworthy and pioneer work, Martin Billeter and
colleagues cloned an infectious cDNA corresponding to the
antigenome of Edmonston MV and established an original and
efficient reverse genetics procedure to rescue the corresponding
virus (Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M.
Huber, K. Dotsch, G. Christiansen, and M. Billeter., 1995. Rescue
of measles viruses from cloned DNA. EMBO Journal. 14:5773-5784) and
WO 97/06270. They developed an Edmonston vector for the expression
of foreign genes (Radecke, F., and M. Billeter. 1997. Reverse
genetics meets the nonsegmented negative-strand RNA viruses.
Reviews in Medical Virology. 7:49-63.) and demonstrated its large
capacity of insertion (as much as 5 kb) and its high stability at
expressing transgenes (Singh, M., and M. Billeter. 1999. A
recombinant measles virus expressing biologically active human
interleukin-12. J. Gen. Virol 80:101-106; Singh, M., R. Cattaneo,
and M. Billeter. 1999. A recombinant measles virus expressing
hepatitis B virus surface antigen induces humoral immune responses
in genetically modified mice. J. Virol. 73:4823-4828; Spielhofer,
P., T. Bachi, T. Fehr, G. Christiansen, R. Cattaneo, K. Kaelin, M.
Billeter, and H. Naim. 1998 Chimeric measles viruses with a foreign
envelope. J. Virol. 72:2150-2159); Wang, Z, T. Hangartner, L. Comu,
A. Martin, M. Zuniga, M. Billeter, and H. Naim. 2001. Recombinant
measles viruses expressing heterologus antigens of mumps and simian
immunodeficiency viruses. Vaccine. 19:2329-2336. This vector was
cloned from the Edmonston B strain of MV propagated in HeLa cells
(Ballart, I., D. Eschle, R. Cattaneo, A. Schmid, M. Metzler, J.
Chan, S. Piflko-Hirst, S. A. Udem, and M. A. Billeter. 1990.
Infectious measles virus from cloned cDNA. Embo J. 9:379-384).
[0007] In addition, recombinant measles virus expressing Hepatitis
B virus surface antigen has been produced and shown to induce
humoral immune responses in genetically modified mice (Singh M. R.
et al, 1999, J. virol 73: 4823-4828).
[0008] MV vaccine induces a very efficient, life-long immunity
after a single low-dose injection (10.sup.4 TCID.sub.50) (33,34).
Protection is mediated both by antibodies and by CD4+ and CD8+ T
cells. The MV genome is very stable and reversion to pathogenicitiy
has never been observed with this vaccine. MV replicates
exclusively in the cytoplasm, ruling out the possibility of
integration in host DNA. Furthermore, an infectious cDNA clone
corresponding to the anti-genome of the Edmonston strain of MV and
a procedure to rescue the corresponding virus have been established
(35). This cDNA has been made into a vector to express foreign
genes (36). It can accommodate up to 5 kb of foreign DNA and is
genetically very stable (37, 38, 39).
[0009] From the observation that the properties of the measles
virus and especially its ability to elicit high titers of
neutralizing antibodies in vivo and its property to be a potent
inducer of long lasting cellular immune response, the inventors
have proposed that it may be a good candidate for the preparation
of compositions comprising recombinant infectious viruses
expressing antigenic peptides or polypeptides of determined RNA
viruses, including especially retroviruses or flaviviruses, to
induce neutralizing antibodies against said RNA virus and
especially said retroviruses or flaviviruses which preferably could
be suitable to achieve at least some degree of protection against
said RNA viruses, especially retroviruses or flaviviruses, in
animals and more preferably in human hosts. Especially, MV strains
and in particular vaccine strains have been elected in the present
invention as candidate vectors to induce immunity against both
measles virus and RNA virus whose constituent is expressed in the
designed recombinant MV, in exposed infant populations because they
are having no. MV immunity. Adult populations, even already MV
immunized individuals, may however also benefit from MV recombinant
immunization because re-administering MV virus under the
recombinant form of the present invention may result in a boost of
anti-MV antibodies.
[0010] Among retroviruses of interest, the inventors have chosen
AIDS retroviruses, including HIV-1 and among flaviviruses, some
which are important human pathogens such as Yellow Fever Virus
(YFV) and West Nile Virus (WNV).
[0011] The YFV and WNV belong to the family Flavivindae described
in Fields virology (3 edition, vol. 1, 1996, Lippincott--Raven
publishers--Fields B N et al).
[0012] The invention relates to a ecombinant mononegavirales virus
expressing a heterologous amino acid sequence, said recombinant
virus being capable of eliciting a humoral and/or a cellular immune
response against said heterologous amino acid sequence including in
individuals having preexisting measles virus immunity.
[0013] In a first embodiment, the invention especially provides
recombinant measles viruses capable of expressing antigens and
especially epitopes derived from antigens of RNA viruses including
retroviruses or flaviviruses.
[0014] The invention also relates to nucleic acid constructs
especially to recombinant nucleic acid constructs expressing the
recombinant measles viruses and expressing therewith antigens or
epitopes of antigens of retroviruses or flaviviruses.
[0015] The invention concerns also processes for the preparation of
such recombinant measles viruses and especially relates to the
production of such recombinant MV in rescue systems.
[0016] The invention is also directed to compositions comprising
said recombinant measles viruses as active principles for
protection of hosts, especially human hosts, against diseases
related to infections by said retroviruses, especially by AIDS
retroviruses, or said flaviviruses, especially Yellow Fever Virus
or West Nile Virus.
[0017] Nucleic acid sequences of Measles Viruses have been
disclosed in International Patent Application WO 98/13501,
especially a DNA sequence of 15,894 nucleotides corresponding to a
DNA copy of the positive strand (antigenomic) message sense RNA of
various wild-type of vaccine measles strains, including Edmonston
Wild-type strain, Moraten strain and Schwarz strain which is
identical to the Moraten strain except for nucleotide positions
4917 and 4924 where Schwarz strain has a <<C>> instead
of a <<T>>.
[0018] In order to produce recombinant measles viruses, a rescue
system has been developed for the Edmonston MV strain and described
in International Patent Application WO 97/06270. The description of
said rescue system contained in WO 97/06270 is incorporated
herewith by reference, and reference is made especially to the
examples of this International application, including the Examples
related to cells and viruses, to generation of cell line 293-346,
plasmid constructions, transfection of plasmids and harvest of
reporter gene products, experimental set-up to rescue MV, helper
cells stably expressing MV N and P proteins as well as T7 RNA
polymerase, MV rescue using helper cells 293-346 and
characterization of rescued MV.
[0019] The rescue system disclosed in WO 97/06270 has been further
developed to include a heat-shock step described in Parks C. L. et
al, 1999, J. virol 73: 3560-3566. The disclosure of this enhanced
measles virus cDNA rescue system is incorporated herewith by
reference.
[0020] The invention thus relates to recombinant measles viruses
expressing a heterologous amino acid sequence derived from an
antigen of a determined RNA virus, especially from a retrovirus or
flavivirus, wherein said recombinant measles virus is capable of
eliciting a humoral and/or a cellular immune response against
measles virus or against said RNA virus, especially retrovirus or
flavivirus or against both measles virus and against said RNA
virus, especially retrovirus or flavivirus. The expression
<<heterologous amino acid sequence>> is directed to an
amino acid sequence which is not derived from the antigens of
measles viruses, said heterologous amino acid sequence being
accordingly derived from a RNA virus, especially from a retrovirus
or flavivirus of interest in order to establish an immune response
in a host, especially in a human and preferably to establish
protection against an infection by said RNA virus, especially
retrovirus or flavivirus.
[0021] The heterologous amino acid sequence expressed in
recombinant measles viruses according to the invention is such that
it is capable of eliciting a humoral and/or cellular immune
response in a determined host, against the RNA virus, especially
retrovirus or flavivirus from which it originates. Accordingly,
this amino acid sequence is one which comprises at least one
epitope of an antigen, especially a conserved epitope, which
epitope is exposed naturally on the antigen or is obtained or
exposed as a result of a mutation or modification or combination of
antigens.
[0022] Antigens used for the preparation of the recombinant measles
viruses are especially envelope antigens of RNA viruses such as
retroviruses or flaviviruses, especially from envelopes of AIDS
viruses including HIV-1 or from envelopes of the Yellow Fever Virus
or envelopes from the West Nile Virus. Other retroviral or
flaviviral antigens may however be advantageously used in order to
derive recombinant measles viruses capable of eliciting antibodies
against said retroviruses or flaviviruses, and the invention
relates in a particular embodiment to antigens from which amino
acid sequences can be derived which elicit the production of
neutralizing antibodies against the retrovirus or flavivirus.
According to another embodiment of the invention, amino acid
sequence of these antigens alternatively or additionally also
elicits a cellular immune response against the retrovirus or
flaviviruses.
[0023] Advantageously, the recombinant measles virus of the
invention also elicits a humoral and/or cellular immune response
against measles virus. This response is however not mandatory
provided the immune response against the RNA virus, especially
retrovirus or flavivirus is indeed obtained.
[0024] According to a preferred embodiment of the invention, the
recombinant measles virus of the invention is obtained within a
rescue system for the preparation of infectious measles viruses.
Accordingly, the recombinant measles virus is a rescued infectious
measles virus recovered from a rescue system.
[0025] A particular recombinant measles virus of the invention is
derived from the Edmonston strain of measles virus.
[0026] Another particular and preferred recombinant measles virus
according to the invention is derived from the--Schwarz strain and
especially from an approved vaccine Schwarz strain such as that
produced under the trademark Rouvax, available from Aventis Pasteur
(France).
[0027] The invention thus provides for a recombinant measles virus
which is recovered from helper cells transfected with a cDNA
encoding the antigenomic RNA ((+)strand) of the measles virus, said
cDNA being recombined with a nucleotide sequence encoding the RNA
viral, especially retroviral or flaviviral, heterologous amino acid
sequence.
[0028] The expression <<encoding>> in the above
definition encompasses the capacity of the cDNA to allow
transcription of a full length antigenomic (+)RNA, said cDNA
serving especially as template for transcription. Accordingly, when
the cDNA is a double stranded molecule, one of the strands has the
same nucleotide sequence as the antigenomic (+) strand RNA of the
measles virus, except that <<U>> nucleotides are
substituted by <<T>> in the cDNA. Such a cDNA is for
example the insert corresponding to the measles virus, contained in
the pTM-MVSchw plasmid deposited under No I-2889 at the CNCM Paris,
France) on Jun. 12, 2002. This plasmid is represented on FIG.
2A.
[0029] The expression "cDNA" used for the description of the
nucleotide sequence of the molecule of the invention merely relates
to the fact that originally said molecule is obtained by reverse
transcription of the full length genomic (-)RNA genome of viral
particles of the measles virus.
[0030] This should not be regarded as a limitation for the methods
used for its preparation. The invention thus encompasses, within
the expression "cDNA", every DNA provided it has the above defined
nucleotide sequence. Purified nucleic acids, including DNA are thus
encompassed within the meaning cDNA according to the invention,
provided said nucleic acid, especially DNA fulfils the above-given
definitions.
[0031] The helper cells according to the rescue system are
transfected with a transcription vector comprising the cDNA
encoding the full length antigenomic (+)RNA of the measles virus,
when said cDNA has been recombined with a nucleotide sequence
encoding the heterologous amino acid sequence of interest
(heterologous nucleotide sequence) and said helper cells are
further transfected with an expression vector or several expression
vectors providing the helper functions including those enabling
expression of trans-acting proteins of measles virus, i.e., N, P
and L proteins and providing expression of an RNA polymerase to
enable transcription of the recombinant cDNA and replication of the
corresponding viral RNA.
[0032] The invention relates in particular to the preparation of
recombinant measles viruses bearing epitopes of antigens of HIV
retroviruses. It encompasses especially a recombinant measles virus
expressing a heterologous amino acid sequence which is derived from
an envelope antigen of HIV and which is especially derived from an
envelope protein or glycoprotein of HIV-1.
[0033] The antigens of interest in this respect are especially
gp160, gp120 and gp41 of HIV-1 or gp140, GAG or TAT of HIV-1.
[0034] In a particular embodiment of the invention, the
heterologous amino acid sequence is derived from a recombinant
gp160, gp120 of HIV-1 or gp140, GAG or TAT of HIV-1.
[0035] The invention is directed in particular to a recombinant
measles virus wherein the V1, V2 and/or V3 loops of the gp120 (or
gp160) antigen are deleted or deleted in part, individually or in
combination in such a way that conserved epitopes are exposed on
the obtained recombinant gp120 antigen.
[0036] The V1, V2 and V3 loops of the gp120 (or gp160) antigen of
HIV-1 have been especially disclosed in Fields virology (Fields B.
N. et al--Lippincoft Raven publishers 1996, p. 1953-1977).
[0037] According to another embodiment of the invention, the
recombinant measles virus is such that it expresses a heterologous
amino acid sequence derived from the gp120 (or gp160) antigen of
HIV-1, wherein the V1, V2 and/or V3 loops of the gp120 (or gp160)
antigen are substituted or substituted in part, individually or in
combination, in such a way that conserved epitopes are exposed on
the obtained recombinant gp120 (or gp160) antigen.
[0038] According to another particular embodiment, the recombinant
measles virus expressing a heterologous DNA sequence derived from
an envelope antigen of HIV-1 is derived from the gp120 antigen in
such a way that the V1 and V2 loops are deleted and the V3 loop is
substituted for the sequence AAELDKWASAA.
[0039] According to another particular embodiment of the invention,
the recombinant measles virus is one expressing an heterologous
amino acid sequence selected among gp160.DELTA.V3,
gp160.DELTA.V1V2; gp160.DELTA.V1V2V3, gp140.DELTA.V3,
gp140.DELTA.V1V2, gp140.DELTA.V1V2V3, which heterologous amino acid
sequences are schematically represented on FIG. 1.
[0040] The invention also relates to recombinant measles viruses as
defined according to the above statements, wherein the amino acid
sequence is derived from an antigen of the Yellow Fever virus
selected among the envelope (Env) or the NS1 proteins or
immunogenic mutants thereof.
[0041] The invention also relates to recombinant measles viruses as
defined according to the above statements, wherein the amino acid
sequence is derived from an antigen of the West Nile virus selected
among the envelope (E), premembrane (preM) or immunogenic mutants
thereof.
[0042] The invention also relates to recombinant measles viruses or
to virus like particles (VLP) which express double or multiple
recombinant antigens, especially multiple HIV antigens (including
fragments thereof) or flavivirus antigens, against which an immune
response is sought. Such recombinant measles viruses or VLP may
advantageously express antigens from different viruses and thus
provide immunogens against various viruses.
[0043] The invention further relates to recombinant measles viruses
according to anyone of the above definitions, wherein the cDNA
required for the expression of the viral particles, which is
comprised within the EdB-tag virus vector or preferably within the
pTM-MVSchw vector is recombined with the ATU sequence of FIG. 8 or
of the ATU sequence illustrated in FIG. 2C, said ATU being inserted
in a position of the EdB-tag vector or of the pTM-MVSchw vector
taking advantage of the gradient of the viral genome to allow
various levels of expression of the transgenic sequence encoding
the heterologous amino acid sequence inserted in said ATU. The
invention advantageously enables the insertion of such heterologous
DNA sequences in a sequence which is designated an Additional
Transcription Unit (ATU) especially an ATU as disclosed by Billeter
et al in WO 97/06270.
[0044] The advantageous immunological properties of the recombinant
measles viruses according to the invention can be shown in an
animal model which is chosen among animals susceptible to measles
viruses and wherein the humoral and/or cellular immune response
against the heterologous antigen and/or against the measles virus
is determined.
[0045] Among such animals suitable to be used as model for the
characterization of the immune response, the skilled person can
especially use mice and especially recombinant mice susceptible to
measles viruses, or in monkeys.
[0046] In a preferred embodiment of the invention, the recombinant
measles virus of the invention is suitable to elicit neutralizing
antibodies against the heterologous amino acid sequence in a
mammalian animal model susceptible to measles virus. Especially,
this immune response comprising elicitation of neutralizing
antibodies can be sought in recombinant mice or monkeys.
[0047] According to another particular embodiment of the invention,
especially when the heterologous amino acid sequence is derived
from one of the envelope proteins of HIV-1 and where it elicits
antibodies capable of neutralizing a primary HIV isolate, the
response is advantageously tested on indicater cells such as
P4-CCR5 cells available from the NIH (NIH AIDS Research and
Reference Reagent Program). (Chameau P. et al--1994--J. Mol. Biol.
241: 651-662).
[0048] According to another preferred embodiment, the recombinant
measles virus according to the invention elicits neutralizing
antibodies against the heterologous amino acid sequence in a
mammal, with a titre of at least 1/40000 when measured in ELISA,
and a neutralizing titre of at least 1/20.
[0049] The invention also relates to a recombinant measles virus
nucleotide sequence comprising a replicon comprising (i) a cDNA
sequence encoding the full length antigenomic (+)RNA of measles
virus operatively linked to (ii) an expression control sequence and
(iii) a heterologous DNA sequence coding for a determined
heterologous amino acid sequence, said heterologous DNA sequence
being cloned in said replicon in conditions allowing its expression
and in conditions not interfering with transcription and
replication of said cDNA sequence, said replicon having a total
number of nucleotides which is a mutiple of six.
[0050] A particular cDNA sequence is the sequence of the cDNA of
the Schwarz strain depicted on FIG. 11. Such a cDNA can be obtained
from pTM-MVSchw.
[0051] pTM-MVSchw is a plasmid derived from Bluescript containing
the complete sequence of the measles virus, vaccine strain Schwarz,
under the control of the promoter of the T7 RNA polymerase. Its
size is 18967 nt.
[0052] The invention concerns also a recombinant measles virus
vector comprising the above defined recombinant measles virus
nucleotide sequence.
[0053] The <<rule of six>> is expressed in the fact
that the total number of nucleotides present in the recombinant
cDNA resulting from recombination of the cDNA sequence derived from
reverse transcription of the antigenomic RNA of measles virus, and
the heterologous DNA sequence finally amount to a total number of
nucleotides which is a multiple of six, a rule which allows
efficient replication of genome RNA of the measles virus.
[0054] A preferred recombinant measles virus vector according to
the above definition is such that the heterologous DNA virus vector
wherein the heterologous DNA sequence is cloned within an
Additional Transcription Unit (ATU) inserted in the cDNA
corresponding to the antigenomic RNA of measles virus.
[0055] The additional transcription unit (ATU) is disclosed on FIG.
2A; it can be modified provided it ultimately enables the obtained
replicon in the vector to comply with the rule of six.
[0056] The location of the ATU within the cDNA derived from the
antigenomic RNA of the measles virus can vary along said cDNA. It
is however located in such a site that it will benefit from the
expression gradient of the measles virus.
[0057] This gradient corresponds to the mRNA abundance according to
the position of the gene relative to the 3' end of the template.
Accordingly, when the polymerase operates on the template (either
genomic and anti-genomic RNA or corresponding cDNAs), it
synthetizes more RNA made from upstream genes than from downstream
genes. This gradient of mRNA abondance is however relatively smooth
for measles virus. Therefore, the ATU or any insertion site
suitable for cloning of the heterologous DNA sequence can be spread
along the cDNA, with a preferred embodiment for an insertion site
and especially in an ATU, present in the N-terminal portion of the
sequence and especially within the region upstream from the L-gene
of the measles virus and advantageously upstream from the M gene of
said virus and more preferably upstream from the N gene of said
virus.
[0058] Depending on the expression site and the expression control
of the heterologous DNA, the vector of the invention allows the
expression of the heterologous amino acid sequence as a fusion
protein with one of the measles virus proteins.
[0059] Alternatively, the insertion site of the DNA sequence in the
cDNA of the measles virus can be chosen in such a way that the
heterologous DNA expresses the heterologous amino acid sequence in
a form which is not a fusion protein with one of the proteins of
the measles virus.
[0060] The recombinant measles virus vector according to any of the
preferred definitions contains advantageously a heterologous DNA
sequence which encodes a retroviral, a flaviviral amino acid
sequence.
[0061] As an example, this amino acid sequence is derived from an
antigen of a retrovirus selected among HIV retroviruses, or a
flavivirus, especially the Yellow Fever virus or the West Nile
virus.
[0062] In a particular embodiment of the invention, the
heterologous amino acid sequence encoded by the recombinant measles
virus vector is derived from an envelope antigen of an HIV
retrovirus, especially from HIV-1.
[0063] In a preferred embodiment, this amino acid sequence encoded
by the heterologous DNA sequence is selected among the gp160, the
gp120 or gp41 of HIV-1, or the gp140 of HIV-1, or a mutated version
of said antigens.
[0064] As one result which is expected by expressing the
recombinant measles virus vector of the invention is the
elicitation of an immune response, especially a humoral and/or
cellular immune response, against the heterologous amino acid
sequence encoded by the vector, it is preferred that the
heterologous DNA sequence used is one which codes for an antigen or
a mutated antigen which enables exposition of neutralizing epitopes
on the produced expression product of said vector.
[0065] In a particular embodiment, the heterologous amino acid
sequence expressed, can expose epitopes which are not accessible or
not formed in the native antigen from which the heterologous amino
acid sequence derives.
[0066] In a preferred embodiment of the invention, the heterologous
DNA sequence encodes gp160.DELTA.V3, gp160.DELTA.V1V2,
gp160.DELTA.V1V2V3, gp140.DELTA.V3, gp140.DELTA.V1V2,
gp140.DELTA.V1V2V3.
[0067] Heterologous amino acid sequences are especially disclosed
on FIG. 1 and can be prepared according to well-known methods
starting from sequences of antigens or corresponding DNA sequences
of said antigens obtained from various HIV-1 isolates.
[0068] According to a preferred embodiment of the invention, the
recombinant measles virus vector is designed in such a way that the
particles produced in helper cells transfected or transformed with
said vector containing the DNA encoding the full length antigenomic
(+)RNA of measles virus, originated from a measles virus strain
adapted for vaccination, enable the production of viral particles
for use in immunogenic compositions, preferably protective or even
vaccine compositions.
[0069] Among measles virus strains adapted for vaccination, one can
cite the Edmonston B. strain and the Schwarz strain, the latter
being preferred and distributed by the company Aventis Pasteur
(Lyon France) as an approved vaccination strain of measles
virus.
[0070] The nucleotide sequences of the Edmonston B. strain and of
the Schwarz strain, have been disclosed in WO 98/13505.
[0071] In order to prepare the recombinant measles virus vector of
the invention, the inventors have designed plasmid pTM-MVSchw which
contains the cDNA resulting from reverse transcription of the
antigenomic RNA of measles virus and an adapted expression control
sequence including a promoter and terminator for the T7
polymerase.
[0072] The recombinant measles virus vector according to the
invention is preferably a plasmid.
[0073] Preferred vectors are those obtained with the nucleotide
sequence of the Edmonston B. strain deposited on Jun. 12, 2002
especially: TABLE-US-00001 pMV2(EdB)gp160[delta]V3HIV89.6P CNCM
I-2883 pMV2(EdB)gp160HIV89.6P CNCM I-2884 pMV2(EdB)gp140HIV89.6P
CNCM I-2885 pMV3(EdB)gp140[delta]V3HIV89.6P CNCM I-2886
pMV2(EdB)-NS1YFV17D CNCM I-2887 pMV2(EdB)-EnvYFV17D CNCM
I-2888.
[0074] Other preferred vectors are those obtained with the
nucleotide sequence of the Schwarz strain, deposited at the CNCM on
May 26, 2003: TABLE-US-00002 pTM-MVSchw2-Es(WNV) CNCM I-3033
pTM-MVSchw2-GFPbis- CNCM I-3034 pTM-MVSchw2-p17p24[delta]myr(HIVB)
CNCM I-3035 pTM-MVSchw3-Tat(HIV89-6p) CNCM I-3036 pTM-MVschw3-GFP
CNCM I-3037 pTM-MVSchw2-Es (YFV) CNCM I-3038 and
[0075] the vectors deposited at the CNCM on Jun. 19, 2003:
TABLE-US-00003 pTM-MVSchw2-gp140 [delta] V1 V2 V3 (HIV89-6) CNCM
I-3054 pTM-MVSchw2-gp140 [delta] V3 (HIV89-6) CNCM I-3055
pTM-MVSchw2-gp160 [delta] V1 V2 V3 (HIV89-6) CNCM I-3056
pTM-MVSchw2-gp160 [delta] V1 V2 (HIV89-6) CNCM I-3057
pTM-MVSchw2-Gag SIV239 p17-p24 [delta] CNCM I-3058 myr-3-gp140
(HIV89-6)
[0076] I-2883 (pMV2(EdB)gp160[delta]V3HIV89.6P) is a plasmid
derived from Bluescript containing the complete sequence of the
measles virus (Edmonston strain B), under the control of the T7 RNA
polymerase promoter and containing the gene of the
gp160.DELTA.V3+ELDKWAS of the virus SVIH strain 89.6P inserted in
an ATU at position 2 (between the N and P genes of measles virus).
The size of the plasmid is 21264 nt.
[0077] I-2884 (pMV2(EdB)gp160HIV89.6P) is a plasmid derived from
Bluescript containing the complete sequence of the measles virus
(Edmonston strain B), under the control of the T7 RNA polymerase
promoter and containing the gene of the gp160 of the SVIH virus
strain 89.6P inserted in an ATU at position 2 (between the N and P
genes of measles virus). The size of the plasmid is 21658 nt.
[0078] I-2885 (pMV2(EdB)gp140HIV89.6P) is a plasmid derived from
Bluescript containing the complete sequence of the measles virus
(Edmonston strain B), under the control of the T7 RNA polymerase
promoter and containing the gene of the gp140 of the SVIH virus
strain 89.6P inserted in an ATU at position 2 (between the N and P
genes of measles virus). The size of the plasmid is 21094 nt.
[0079] I-2886 (pMV3(EdB)gp140[delta]V3HIV89.6P) is a plasmid
derived from Bluescript containing the complete sequence of the
measles virus (Edmonston strain B), under the control of the T7 RNA
polymerase promoter and containing the gene of the
gp140.DELTA.V3(ELDKWAS) of the SVIH virus strain 89.6P inserted in
an ATU at position 2 (between the N and P genes of measles virus).
The size of the plasmid is 21058 nt.
[0080] I-2887 (pMV2(EdB)-NS1YFV17D) is a plasmid derived from
Bluescript containing the complete sequence of the measles virus
(Edmonston strain B), under the control of the T7 RNA polymerase
promoter and containing the NS1 gene of the Yellow Fever virus (YFV
17D) inserted in an ATU at position 2 (between the N and P genes of
measles virus). The size of the plasmid is 20163 nt.
[0081] I-2888 (pMV2(EdB)-EnvYFV17D) is a plasmid derived from
Bluescript containing the complete sequence of the measles virus
(Edmonston strain B), under the control of the T7 RNA polymerase
promoter and containing the Env gene of the Yellow Fever virus (YFV
17D) inserted in an ATU at position 2 (between the N and P genes of
measles virus). The size of the plasmid is 20505 nt.
[0082] I-3033 (pTM-MVSchw2-Es(WNV) is a plasmid derived from
Bluescript containing a cDNA sequence of the complete infectious
genome of the measles virus (Schwarz strain), under the control of
the T7 RNA polymerase promoter and expressing the gene of the
secreted envelope, (E) of the West Nile virus (WNV), inserted in an
ATU.
[0083] I-3034 (pTM-MVSchw2-GFPbis) is a plasmid derived from
Bluescript containing a cDNA sequence of the complete infectious
genome of the measles virus (Schwarz strain), under the control of
the T7 RNA polymerase promoter and expressing the gene of the GFP
inserted in an ATU.
[0084] I-3035 (pTM-MVSchw2-p17p24[delta]myr(HIVB) is a plasmid
derived from Bluescript containing a cDNA sequence of the complete
infectious genome of the measles virus (Schwarz strain), under the
control of the T7 RNA polymerase promoter and expressing the gene
of the gag gene encoding p17p24.DELTA.myrproteins of the HIVB virus
inserted in an ATU.
[0085] I-3036 (pTMVSchw3-Tat(HIV89-6p) is a plasmid derived from
Bluescript containing a cDNA sequence of the complete infectious
genome of the measles virus (Schwarz strain), under the control of
the T7 RNA polymerase promoter and expressing the gene of the Tat
gene of the virus strain 89.6P inserted in an ATU.
[0086] I-3037 (pTM-MVSchw3-GFP) is a plasmid derived from
Bluescript containing a cDNA sequence of the complete infectious
genome of the measles virus (Schwarz strain) under the control of
the T7 RNA polymerase promoter and expressing the gene of the GFP
gene inserted in an ATU having a deletion of one nucleotide.
[0087] I-3038 (pTM-MVSchw2-Es) (YFV) is a plasmid derived from
Bluescript containing a cDNA sequence of the complete infectious
genome of the measles virus (Schwarz strain) under the control of
the T7 RNA polymerase promoter and expressing the gene of the
secreted protein of the Fever virus (YFV) inserted in an ATU.
[0088] I-3054 (pTM-MVSchw2-gp140[delta] V1 V2 V3 (HIV89-6)) is a
plasmid derived from Bluescript containing a cDNA sequence of the
complete infectious genome of the measles virus (Schwarz strain),
under the control of the T7 RNA polymerase promoter and expressing
the gene encoding gp140 [delta] V1 V2 (HIV 89-6) inserted in an
ATU.
[0089] I-3055 (pTM-MVSchw2-gp140 [delta] V3 (HIV89-6)) is a plasmid
derived from Bluescript containing a cDNA sequence of the complete
infectious genome of the measles virus (Schwarz strain), under the
control of the T7 RNA polymerase promoter and expressing the gene
encoding gp14 [delta] V3 (HIV 89-6) inserted in an ATU.
[0090] I-3056 (pTM-MVSchw2-gp160 [delta] V1 V2 V3 (HIV89-6)) is a
plasmid derived from Bluescript containing a cDNA sequence of the
complete infectious genome of the measles virus (Schwarz strain),
under the control of the T7 RNA polymerase promoter and expressing
the gene encoding gp160 [delta] V1 V2 V3 (HIV 89-6) inserted in an
ATU.
[0091] I-3057 (pTM-MVSchw2-gp160 [delta] V1 V2 (HIV89-6)) is a
plasmid derived from Bluescript containing a cDNA sequence of the
complete infectious genome of the measles virus (Schwarz strain),
under the control of the T7 RNA polymerase promoter and expressing
the gene encoding gp160 [delta] V1 V2 (HIV 89-6) inserted in an
ATU.
[0092] I-3058 (pTM-MVSchw2-Gag SIV239 p17-p24 [delta] myr-3-gp140
(HIV89-6)) is a plasmid derived from Bluescript containing a cDNA
sequence of the complete infectious genome of the measles virus
(Schwarz strain), under the control of the T7 RNA polymerase
promoter and expressing the gene encoding Gag SIV239 p17-p24
[delta] myr-3-gp140 (HIV89-6) inserted in an ATU.
[0093] In a particular embodiment of the invention, the replicon
contained in the recombinant measles virus vector is designed
according to the map of FIG. 2 wherein <<insert>>
represents the heterologous DNA sequence.
[0094] When the heterologous DNA sequence present in the
recombinant measles virus vector of the invention is derived from
the Yellow Fever Virus (YFV), it is advantageously selected among
YFV 17D 204 commercialized by Aventis Pasteur under the trademark
Stamaril.RTM..
[0095] When the heterologous DNA sequence present in the
recombinant measles virus vector of the invention is derived from
the West Nile Virus (WNV), it is advantageously selected among the
neurovirulente strain IS 98-ST1.
[0096] The invention also relates to a rescue system for the
assembly of recombinant measles virus expressing a heterologous
amino acid sequence, which comprises a determined helper cell
recombined with at least one vector suitable for expression of T7
RNA polymerase and expression of the N, P and L proteins of the
measles virus transfected with a recombinant measles virus vector
according to anyone of the definitions provided above.
[0097] The recombinant viruses of the invention or the VLP can also
be produced in vivo by a live attenuated vaccine like MV.
[0098] The recombinant viruses of the invention or the VLP can be
used in immunogenic compositions or in vaccine compositions, for
the protection against RNA viruses, which antigens are expressed in
the recombinant virus or in the VLP, as disclosed above and
illustrated in the following examples.
[0099] The invention especially provides for immunogenic
compositions or for vaccine compositions useful against HIV virus,
West Nile virus or Yellow Fever virus.
[0100] The invention also concerns the use of the recombinant
viruses disclosed or of the VLP, or of the recombinant vectors, for
the preparation of immunogenic compositions or for the preparation
of vaccine compositions.
[0101] The invention also relates to antibodies prepared against
said recombinant viruses or against said VLP, especially to
protective antibodies and to neutralizing antibodies. Antibodies
may be polyclonal antibodies, or monoclonal antibodies.
[0102] The recombinant viruses of the invention or the VLP can be
associated with any appropriate adjuvant, or vehicle which may be
useful for the preparation of immunogenic compositions.
[0103] Various aspects of the invention will appear in the examples
which follow and in the drawings.
LEGEND OF THE FIGURES
[0104] FIG. 1. HIV1 Env glycoprotein constructions. (A) gp160
constructions full-length and .DELTA.V3-MELDKWASM, .DELTA.V1V2 and
.DELTA.V1V2V3 mutants (from top to bottom). The BbsI and MfeI
restriction sites used to introduce the .DELTA.V3 deletion in the
other constructions are indicated. (B) gp140 constructions are the
same as gp160 except that the intracytoplasmic and transmembrane
regions of the gp41 have been deleted.
[0105] FIG. 2A. Schematic map of the pTM-MV Schw plasmid. To
construct the complete sequence, the six fragments represented in
the upper part were generated and recombined step by step using the
unique restriction sites indicated. T7=T7 promoter; hh=hammerhead
ribozyme; h.DELTA.v=hepatitis delta ribozyme (=.delta.); T7t=T7 RNA
polymerase terminator.
[0106] FIG. 2B. The pMV(+) vectors with ATU containing a green
fluorescent protein (GFP) gene in position 2 and position 3. The MV
genes are indicated: N (nucleoprotein), PVC (phosphoprotein and V C
proteins), M (matrix), F (fusion), H (hemaglutinin), L
(polymerase). T7: T7 RNA polymerase promoter; T7t: 17 RNA
polymerase terminator; .delta.: hepatitis delta virus (HDV)
ribozyme; ATU: additional transcription unit.
[0107] FIG. 2C. ATU sequence: small letters represent additional
sequences (copy of the N-P intergenic region of measles virus) plus
cloning sites. Capital letters correspond to the inserted enhanced
GFP sequence. This sequence is inserted at the SpeI site (position
3373) of the cDNA sequence of the Schwarz strain of the measles
virus for ATU2 and at the SpeI site (position 9174) for the ATU3.
The mutation which distinguishes normal ATU from bis (in
pTM-MVSchw2-gfp and pTM-MVSchw2-GFPbis) is a substituted C (Capital
letter) at the end of ATU.
[0108] FIG. 3 (A): shows that ENV.sub.HIV89.6 expression was
similar for passages 2 and 5, confirming the stability of
expression of transgenes in this system.
[0109] FIG. 3 (B): construction of Schwarz measles viruses (MVSchw)
expressing HIV-1 antigens. (1-3054 to 1-3058). Expression of HIV
antigens in recombinant pTM-MVSchw.
[0110] FIG. 3BA: Expression of HIV-1 envelope glycoproteins in
recombinant pTM-MVSchw. Vero cells were infected with the different
recombinant viruses for 48H and expression of HIV Env was
determined by western blot. 30 .mu.g of each cell lysate were
resolved on 4-12% SDS-PAGE, blotted onto nitrocellulose membranes
and probed with a mouse monoclonal anti-HIV gp.sup.120 (Chessie,
NIH) antibody. Anti-mouse IgG RPO conjugate was used as second
antibody and proteins were detected using an ECL detection kit.
[0111] FIG. 3BB: (1) construct of double recombinant
pTM-MVSchw2-Gag-3gp140
[0112] Some recombinant vectors expressing two different
heterologous antigens have been constructed. They were obtained by
ligation of two different recombinant pTM-MVSchw plasmids
containing different inserts in position 2 and position 3. Plasmid
pTM-MVSchw2-Gag-3-gp140 is shown. From this plasmid a recombinant
virus was rescued that expressed both Gag and gp140 proteins (FIG.
3B(2) Western blot). Using appropriate constructions of the
different inserted heterologous genes, such recombinant MV
expressing two heterologous viral proteins may produce
<<virus like particles >> (VLP) assembled in infected
cells and secreted: Gag-Env from retroviruses or prM/E from
flaviviruses. Such VLP are good immunogens. Produced in vivo by a
live attenuated vaccine like MV, they should be even more
immunogenic.
[0113] (2) Expression of HIV-1 gp140 and SIV239 Gag in recombinant
pTM-MVSchw2-Gag.sub.SIV (p17-p24 [delta] myr)-3-gp.sup.140.sub.HIV.
HIV gp140 and SIV Gag were detected in lysates of infected Vero
cells. (A) a mouse monoclonal anti-HIV gp120 and (B) serum from
macaque infected with SIVmac251.
[0114] FIG. 3C: Expression of HIV-1 Gag (p17-p24 .DELTA.myr) in
recombinant pTM-MVSchw2-Gag.sub.HIV (p17-p24 [delta] myr). HIV Gag
were detected in lysates of infected Vero cells with a mouse
monoclonal anti-HIV Gag antibody.
[0115] FIG. 3D: Expression of HIV-1 Tat protein in recombinant
pTM-MVSchw. Vero cells were infected with MVSchw-Tat HIV
recombinant or control MVSchw viruses for 48H and expression of HIV
Tat was determined by western blot. 30 .mu.g of each cell lysate
were resolved on 4-12% SDS-PAGE, blotted onto nitrocellulose
membranes and probed with a mouse monoclonal anti-HIV Tat (BH10,
NIH) antibody. Anti-mouse IgG RPO conjugate was used as second
antibody and proteins were detected using an ECL detection kit.
[0116] FIG. 4. Growth kinetics of recombinant
MV.sub.EdB-Env.sub.HIV viruses on Vero cells. Cells on 35 mm dishes
were infected with recombinant viruses at different MOI (as
indicated). At each time point, cells were collected and
cell-associated virus titers were determined using the TCID.sub.50
method on Vero cells. (A) Infections with MV EdB-tag and different
MV-HIV recombinant viruses at MOI=0.0001. (B) Infections with
MV2gp160.sub.HIV at two different MOI (0.0001 and 0.01).
[0117] FIG. 5. Anti-HIV and anti-MV humoral immune responses in
mice inoculated with recombinant MV.sub.EdB-Env.sub.HIV viruses.
A-B Four groups of 3 mice were immunized with 10.sup.7 TCID.sub.50
of each MV-HIV recombinant virus. Antibody titers against MV (A)
and HIV Env (B) were determined by ELISA in sera collected 28 days
post inoculation. C-F: Anti-HIV and anti-MV antibody titers in
IFNAR.sup.-/-/CD46.sup.+/- mice immunized with MV-Env.sub.HIV
viruses. (C) Anti-MV and anti-HIV titers detected 28 days after
injection of increasing doses of MV.sub.EdB-gp160 (3 mice per
group). (D) Anti-MV (black bars), anti-HIV (gray bars) and
anti-ELDKWAS (white bars) titers detected 28 days after injection
of 5 106 TCID.sub.50 of MV-Env.sub.HIV viruses (6 mice per group).
Results are expressed as the mean values.+-.SD.
[0118] FIG. 6. Neutralizing activities against Bx08 of sera from
mice immunized with MV2-gp140.sub.HIV89.6 and MV2-gp160.sub.HIV89.6
viruses. Primary isolate Bx08 was provided by C. Moog (Strasbourg,
France) and propagated once on PHA-stimulated PBMC to obtain viral
stocks. 2 ng of virus was incubated for 30 min at 37.degree. C.
with 25 .mu.l of each mouse serum (collected one month
post-infection) before infection of P4R5 cells in a 96-well plate.
Cells were then cultured in DMEM containing 10% of fetal calf serum
until 2 days post-infection, at wich time .beta. Galactosidase
activity was measured with a chemiluminescence test (Roche,
Germany). Lane 1: serum of a MV.sub.EdB-Tag immunized mouse; Lane
2: serum of a MV2-gp140.sub.HIV-1 immunized mouse; Lane 3: serum of
a MV2-gp160.sub.HIV-1 immunized mouse; Lane 4: non-infected cells.
All assays were performed in triplicate.
[0119] FIG. 7. Edm-HIV Env vaccine candidate stimulates
env-specific lymphocytes in vivo. Two groups of 3 mice were
inoculated with 10.sup.7 TCID.sub.50 of MV2-gp160.sub.HIV virus,
and euthanized 7 day and one 1 month post inoculation. (A) ELISpot
assays performed with splenocytes from immunized mice. Stimulation
with HIV-gp120 purified protein (black) or irrelevant BSA (white).
(B) Splenocytes collected 7 days after immunization were stimulated
either with medium alone (left panel), HIV gp120 (middle panel) of
EdB-tag virus (right panel). Three-color cytofluorometry detected
both CD8+ (upper panel) and CD4+ (lower panel) lymphocytes
producing .gamma.-IFN after HIV gp120 and measles virus
stimulations. Percentages are given according to the total CD8+
(upper panel) and CD4+ (lower panel) lymphocyte gates
respectively.
[0120] FIG. 7C. D. Anti-MV and anti-HIV antibody titers in mice and
macaques immunized with MV2-gp140HIV89.6 virus months after MV
priming. (C) Mice (3 per group) were vaccinated with 10.sup.5
TCID.sub.50 of EdB-tag MV then inoculated twice with 5 10.sup.6
TCID.sub.50 of MV2-gp140.sub.HIV 89.6 virus as Indicated (arrows).
(D) Cynomolgus macaques (# 432 and 404) were vaccinated with Rouvax
then inoculated twice with 5 10.sup.6 TCID.sub.50 of
MV2(qp140.sub.HIV89.6 virus as indicated (arrows).
[0121] FIG. 8. Schematic representation the additional
transcription unit (ATU) and Schwarz MV vector plasmid. (A)
Cis-acting elements of the ATU inserted in position 2 between
phosphoprotein (P) and matrix (M) MV open reading frames. (B).
Representation of the three positions of ATU insertion in the
Schwarz MV vector plasmid.
[0122] FIG. 9. Expression of YFV proteins by recombinant MV. Vero
cells were infected by recombinant EdB-Env.sub.YFV and
EdB-NS1.sub.YFV MV at an MOI of 0.01. Immunofluorescence was
performed using a mouse polyclonal anti-YFV serum and a Cy3
secondary anti-mouse IgG antibody. All the syncytia observed in
infected Vero cells were positive.
[0123] FIG. 10. YFV challenge. Six 4-weeks old mice were inoculated
with a mixture of EdB-Env.sub.YFV and EdB-NS1.sub.YFV viruses
(10.sup.7 TCID.sub.50) and 6 control mice were inoculated with the
same dose of standard EdB-tag virus. After 1 month, anti-MV
serologies were determined and a similar level of antibodies was
observed in the two groups. Mice were challenged and mortality was
observed.
[0124] FIG. 11. Complete nucleotide sequence of the pTM-MVSchw
plasmid (CNCM I-2889). The sequence can be described as follows
with reference to the position of the nucleotides: TABLE-US-00004
1-8 Notl restriction site 9-28 T7 promoter 29-82 Hammer head
ribozyme 83-15976 MV Schwarz antigenome 15977-16202 HDV ribozyme
and T7 terminator 16203-16210 Notl restriction site 16211-16216
Apal restriction site 16220-16226 Kpnl restriction site 16226-18967
pBluescript KS(+) plasmid (Stratagene)
[0125] FIG. 12:
[0126] The flaviral sequences which have been expressed in MV are
the following
[0127] FIG. 12A: YFV Env seq: This is the Env YFV 17D204 sequence
cloned by the inventors. TABLE-US-00005 pos 1 a 3 START codon pos 4
a 51 Env signal peptide pos 52 a 1455 Env sequence pos 1456 a 1458
STOP codon
[0128] The stop and start codons have been added.
[0129] FIG. 12B: YFV NS1 seq: This is the NS1 YFV 17D204 sequence
cloned by the inventors TABLE-US-00006 pos 1 a 3 START codon pos 4
a 78 NS1 signal peptide pos 79 a 1110 NS1 sequence pos 1111 a 1113
STOP codon
[0130] The stop and start codons have been added.
[0131] FIG. 12C: WNV Env seq: this is the Env WNV sequence cloned
by the inventors. TABLE-US-00007 pos 1 a 3 START codon pos 4 a 51
env signal peptide pos 52 a 1485 Env sequence pos 1486 a 1488 STOP
codon
[0132] The stop and start codons have been added.
[0133] FIG. 12D: WNV NS1 seq: This is the NS1 WNV sequence cloned
by the inventors. TABLE-US-00008 pos 1 a 3 START codon pos 4 a 78
NS1 signal peptide pos 79 a 1104 NS1 sequence pos 1105 a 1107 STOP
codon pos 1108 a 1110 STOP codon (a second is added in order to
respect the rule six.)
[0134] The stop and start codons have been added.
[0135] FIG. 13: Schematic representation of recombinant
pTM-MVSchw-sE.sub.WNV. The MV genes are indicated: N
(nucleoprotein), PVC (phosphoprotein and V, C proteins), M
(matrix), F (fusion), H (hemmaglutinin), L (polymerase). T7: T7 RNA
polymerase promoter; T7t: T7 RNA polymerase terminator; .delta.
hepatitis delta virus (HDV) ribozyme; ATU: additional transcription
unit.
[0136] After rescue, the recombinant virus was grown on Vero cell
monolayers. The procedure used to prepare the recombinant virus was
similar to the standard procedures used to prepare the live
attenuated measles vaccines, except for the lyophilization that was
not used.
[0137] The WNV sE expression in Vero cells infected by the MV-WN sE
virus was verified by using indirect immunofluorescence assay as
shown in FIG. 14.
[0138] FIG. 14: Expression of sE protein from WNV in MV induced
syncytia. Immunofluorescence detection of secreted WNV Env (sE)
protein in syncytia induced by recombinant MV-WN sE in Vero cells.
(A, B) sE protein detected at the external surface all around
recombinant MV-induced syncytia. (C, D) intracellular sE protein in
recombinant MV-induced syncytia.
[0139] FIG. 15: Anti-MV serology 1 month after the first
injection.
[0140] FIG. 16: HIV-1 immunogenic sequences prepared for insertion
in plasmid pTM-MVSchw2 illustrated in Example II.
EXAMPLE I
Recombinant Measles Viruses Expressing the Native Envelope
Glycoprotein of HIV1 Clade B, or Envelopes with Deleted Variable
Loops, Induce Humoral and Cellular Immune Responses
[0141] Preparing a vaccine against HIV with its formidable ability
at evading the host immune responses is certainly a daunting task.
However, what we have learned about the immunopathogenesis of the
infection and results already obtained with animal models indicate
that it may be possible (Mascola, J. R., and G. J. Nabel. 2001.
Vaccines for prevention of HIV-1 disease. Immunology. 13:489-495).
Ideally, a preventive immunization should induce 1) antibodies that
neutralize primary isolates, thereby preventing entry into host
cells, and 2) CTL that eliminate the cells that were nevertheless
infected. Antibodies and CTL should be directed at conserved
epitopes that are critical for viral entry and replication into
host cells.
[0142] Several studies, in particular with various candidate
vaccines, show that a good cellular immune response might be able
to control viral load, although not to eliminate the agent
(Mascola, J. R., and G. J. Nabel. 2001. Vaccines for prevention of
HIV-1 disease. Immunology. 13:489-495). On the other hand humoral
immune responses induced so far by subunit vaccines have been
disappointing, mainly because the antibodies induced did not
neutralize primary isolates of HIV. For example, recombinant
vaccines expressing the SIV Env were able to protect macaques
against an homologous, but not an heterologous, challenge (Hu, S.,
et al 1996. Recombinant subunit vaccines as an approach to study
correlates of protection against primate lentivirus infection.
Immunology Letters. 51:115-119). DNA immunization combined with
boosting with soluble recombinant gp could protect macaques against
an heterologous challenge but only against a strain of SIV
genetically related to the vaccine (Boyer, J. et al 1997.
Protection of chimpanzees from high-dose heterologous HIV-1
challenge by DNA vaccination. Nature Medicine. 3:526-532). More
recently, various <<prime-boost>> regimen, using
combinations of naked DNA and viral vectors such as MVA (Amara, R.
et al. 2001. Control of a mucosal challenge and prevention of AIDS
by a multiprotein DNA/MVA vaccine. Science. 292:69-74) or
Adenovirus (Shiver, J. W, et al 2002. Replication-incompetent
adenoviral vaccine vector elicits effective
anti-immunodeficiency-virus immunity. Nature. 415:331-335), gave
reasonable protection against a challenge with pathogenic
SHIV89.6P. <<Prime-boost>> might not be an absolute
requirement since using recombinant live attenuated polio virus
vaccine protected macaques against an SIV251 challenge (Crotty, S.,
et al 2001. Protection against simian immunodeficiency virus
vaginal challenge by using Sabin poliovirus vectors. J. Virol.
75:7435-7452). It is interesting to note that in all these
experiments, even when the animals were not protected against the
infection, immunization caused a delay in, or even abrogated,
clinical disease.
[0143] As shown by crystallography, the V1 and V2 loops of gp120
mask the CD4 binding site and the V3 loop masks the binding sites
for the CXCR4 and CCR5 co-receptors (Kwong, P. D., et al 2000.
Structures of HIV-1 gp120 envelope glycoproteins from
laboratory-adapted and primary isolates. Structure Fold Des.
8:1329-1339; Kwong, P. D. et al 1998. Structure of an HIV gp120
envelope glycoprotein in complex with the CD4 receptor and a
neutralizing human antibody. Nature. 393:648-659; Kwong, P. D., et
al 2000. Oligomeric modeling and electrostatic analysis of the
gp120 envelope glycoprotein of human immunodeficiency virus. J.
Virol. 74:1961-1972). In spite of this, antibodies against the
gp120 CD4 binding site are present in the sera of HIV seropositive
individuals and are able to neutralize several HIV-1 isolates in in
vitro tests (Burton, D. 1997. A vaccine for HIV type 1: the
antibody perspective. Proceedings of the National Academy of
Sciences of the United States of America. 94:10018-10023; Hoffman,
T L et al., 1999. Stable exposure of the coreceptor-binding site in
a CD4-independent HIV-1 envelope protein. Proc Natl Acad Sci USA.
96:6359-6364). Also, some epitopes which are buried in the 3-D
structure of the glycoprotein but become exposed after binding to
the co-receptor, can induce highly neutralizing antibodies (Muster,
T., et al 1993. A conserved neutralizing epitope on gp41 of human
immunodeficiency virus type 1. J. Virol. 67:6642-6647).
Furthermore, neutralizing monoclonal antibodies have been obtained
from patient's B cells (Parren, P. W., et al 1997. Relevance of the
antibody response against human immunodeficiency virus type 1
envelope to vaccine design; Immunol Lett. 57:105-112). They are
directed at gp41 linear epitopes (2F5) (Muster, T., F. et al 1993.
A conserved neutralizing epitope on gp41 of human immunodeficiency
virus type 1. J. Virol. 67:6642-6647), or at gp120 conformational
epitopes (2G12, 17b, 48 db12) (Thali, M., et al 1993.
Characterization of conserved human immunodeficiency virus type 1
gp120 neutralization epitopes exposed upon gp120-CD4 binding. J.
Virol. 67:3978-3988; Trkola, A., et al. 1996. Human monoclonal
antibody 2G12 defines a distinctive neutralization epitope on the
gp120 glycoprotein of human immunodeficiency virus type 1. J.
Virol. 70:1100-1108). Used in synergy they can neutralize in vitro
several primary isolates (Mascola, J. R. et al 1997. Potent and
synergistic neutralization of human immunodeficiency virus (HIV)
type 1 primary isolates by hyperimmune anti-HIV immunoglobulin
combined with monoclonal antibodies 2F5 and 2G12. J. Virol.
71:7198-7206) and protect macaques against a mucosal challenge with
SHIV (Baba, T. W. et al, 2000. Human neutralizing monoclonal
antibodies of the IgG1 subtype protect against mucosal simian-human
immunodeficiency virus infection. Nat Med. 6:200-206; Mascola, J.
R., et al 1999. Protection of Macaques against pathogenic
simian/human immunodeficiency virus 89.6PD by passive transfer of
neutralizing antibodies. J. Virol. 73:4009-4018; Mascola, J. R., et
al 2000. Protection of macaques against vaginal transmission of a
pathogenic HIV-1/SIV chimeric virus by passive infusion of
neutralizing antibodies. Nat Med. 6:207-210). However in infected
people, all these antibodies are present in very low amounts,
diluted in large quantities of non-neutralizing antibodies directed
mainly at the antigenically variable V1, V2 and V3 gp120 loops.
Therefore, there is hope that if one could induce high levels of
such cross-neutralizing antibodies one may achieve at least some
degree of protection. A major goal is to design a vector that will
favor the production of such neutralizing antibodies.
[0144] For this reason, we engineered mutant gp160 (anchored) and
gp140 (soluble) by deleting the hypervariable V1, V2 and V3 loops
individually or in combination to expose conserved epitopes and
induce antibodies able to neutralize primary isolates. In some of
the constructions, we also replaced the V3 loop by the AAELDKWASAA
sequence, especially ELDKWAS sequence flanked on both sides by two
Alanine to maintain the conformation of this gp41 conserved epitope
normally buried in the native protein but able to induce large
spectrum neutralizing antibodies (Muster, T., Fat al 1993. A
conserved neutralizing epitope on gp41 of human immunodeficiency
virus type 1. J Virol. 67:6642-6647; Binley, J. M., et al 2000. A
recombinant human immunodeficiency virus type 1 envelope
glycoprotein complex stabilized by an intermolecular disulfide bond
between the gp120 and gp41 subunits is an antigenic mimic of the
trimeric virion-associated structure. J. Virol. 74:627-643;
Sanders, R. W, et al 2000. Variable-loop-deleted variants of the
human immunodeficiency virus type 1 envelope glycoprotein can be
stabilized by an intermolecular disulfide bond between the gp120
and gp41 subunits. J. Virol. 74:5091-5100). The normal alpha
helical structure of this peptide should be conserved when exposed
in our constructions at the tip of a deleted V3 loop. These
constructions, in which the "immunological decoys" have been
eliminated and the neutralizing epitopes have been exposed, should
be good candidates for the induction of robust neutralizing
antibody responses.
[0145] The HIV gp constructions were introduced into a measles
vaccine vector because it induces very high titers (1/80,000) of
neutralizing anti-measles antibodies. (This is probably because it
replicates in a large number of cells of different types.) One may
hope, therefore, that the antibody response against the engineered
HIV gps will also be strong. Furthermore, measles vaccine is also a
potent inducer of long lasting cellular responses. The recombinant
vaccines induced cross-neutralizing antibodies as well as cellular
immune responses after a single injection in CD46.sup.+/-
IFN-.alpha./.beta._R.sup.-/- mice. Furthermore, they induced immune
responses against HIV in mice and macaques with a pre-existing
anti-MV immunity.
[0146] Construction of Mutant HIV-1 Envelope Glycoproteins.
[0147] The envelope glycoproteins used in this study (FIG. 1) were
derived from SHIV89.6P, a chimeric simian/human immunodeficiency
virus which contains tat, rev, vpu and env genes of HIV1 in an
SIVmac239 background (Reimann, K. A., et al 1996. A chimeric
simian/human immunodeficiency virus expressing a primary patient
human immunodeficiency virus type 1 isolate env causes an AIDS-like
disease after in vivo passage in rhesus monkeys. J. Virol.
70:6922-6928). The env gene is derived from a cytopathic primary
HIV1 isolate, 89.6, which is tropic for both macrophages and T
cells (Collman, R., et al 1992. An infectious molecular clone of an
unusual macrophage-tropic and highly cytopathic strain of human
immunodeficiency virus type 1. J. Virol. 66:7517-7521). The env
sequence was amplified from the plasmid pSHIV-KB9 (NIH) that was
previously cloned after in vivo passages of the original virus
(Karlsson, G. B., et al 1997. Characterization of molecularly
cloned simian-human immunodeficiency viruses causing rapid CD4+
lymphocyte depletion in rhesus monkeys. J. Virol. 71:4218-4225).
The full-length env (gp160) was amplified by PCR (Pfu polymerase)
using primers that contain unique BsiWI and BssHII sites for
subsequent cloning in measles vector: 160E5
(5'-TATCGTACGATGAGAGTGAAGGAGAAATAT-3') and 160E3
(5'ATAGCGCGCATCACMGAGAGTGAGCTCM-3'). The env sequence corresponding
to the secreted form (gp140) was amplified using primers 160E5 and
140E3 (5'-TATGCGCGCTTATCTTATATACCACAGCCAGT-3'). A start and a stop
codon were added at both ends of the genes as well as several
nucleotides after the stop codon in order to respect the "rule of
six", stipulating that the number of nucleotides of MV genome must
be a multiple of 6 (Calain, P., and L. Roux. 1993. The rule of six,
a basic feature for efficient replication of Sendai virus defective
interfering RNA. J. Virol. 67:4822-4830; Schneider, H., et al 1997.
Recombinant measles viruses defective for RNA editing and V protein
synthesis are viable in cultured cells. Virology. 227:314-322).
Both gp160 and gp140 env fragments were cloned in pCR2.1-TOPO
plasmid (Invitrogen) and sequenced to check that no mutations were
introduced.
[0148] Mutants with loop-deletions were generated by PCR
amplification of two overlapping fragments flanking the sequence to
be deleted and annealing of these fragments by PCR. To replace the
V3 sequence by the AAELDKWASAA sequence containing the gp41 epitope
(Muster, T., F. et al 1993. A conserved neutralizing epitope on
gp41 of human immunodeficiency virus type 1. J. Virol.
67:6642-6647), four primers were designed on both sides of BbsI and
MfeI sites encompassing the V3 sequence: .DELTA.V3A1
(5'-ATAAGACATTCAATGGATCAGGAC-3'), .DELTA.V3A2
(5'TGCCCATTTTATCCAATTCTGCAGCATTGTTGTTGGGTCTTGTACAATT-3'),
.DELTA.V3B1 (5'-GATAAATGGGCAAGTGCTGCAAGACAAGCACATTGTMCATTGT-3'),
and .DELTA.V3B2 (5'-CTACTCCTATTGGTTCAATTCTTA-3'). The underlined
sequences in .DELTA.V3A2 and .DELTA.V3B1 correspond to the
AAELDKWASAA epitope with a 12 nucleotides overlap. PCR
amplifications with primer pairs .DELTA.V3A1/.DELTA.V3A2 and
.DELTA.V3B1/.DELTA.V3B2 produced two fragments of 218 and 499 bp
respectively. After gel purification, these fragments were annealed
together by 15 PCR cycles without primers and amplified with
.DELTA.V3A1/.DELTA.V3B2 primers. The resulting 705 bp fragment was
cloned in pCR2.1-TOPO plasmid and sequenced. After digestion by
BbsI and MfeI, the fragment lacking the sequence encoding the V3
loop (.DELTA.V3-AAELDKWASAA) was purified and introduced in place
of the corresponding fragment in the gp160 and gp140 in pCR2.1-TOPO
plasmids.
[0149] The resulting plasmids were designated pMV2-gp160.DELTA.V3
and pMV2-gp140.DELTA.V3.
[0150] The .DELTA.V1V2 mutants were produced using the same
procedure. Two fragments were amplified on both sides of V1V2 loop
using the following primers: 160E5 (5'-TATCGTACG
ATGAGAGTGMGGAGMATAT-3'), .DELTA.V1V2A1 (5'-ATTTAAAGTAACACAGAGTG
GGGTTAATTT-3'), .DELTA.V1V2B1 (5'-GTTACTTTAAATTGTMCACCTCAGTCATTAC
ACAGGCCTGT-3'), .DELTA.V1V2B2
(5'-TTGCATAAAATGCTCTCCCTGGTCCTATAG-3'). The underlined sequences in
.DELTA.V1V2A1 and .DELTA.V1V2B1 correspond to a 12 nucleotide
overlap generated between the two fragments. PCR amplifications
with primer pairs 160E5/.DELTA.V1V2A1 and
.DELTA.V1V2B1/.DELTA.V1V2B2 produced two fragments of 400 and 366
bp respectively. After gel purification, these fragments were
annealed together by 15 PCR cycles without primers and amplified
with 160E5/.DELTA.V1V2B2 primers. The resulting 766 bp fragment was
cloned in pCR2.1-TOPO plasmid and sequenced. After digestion with
BsiWI (in 160E5 primer) and BbsI, the fragment lacking the sequence
encoding the V1V2 loop was purified and introduced in place of the
corresponding fragment in the gp160 and gp140 in pCR2.1-TOPO
plasmids.
[0151] To obtain the .DELTA.V1V2V3 mutants, the BsiWI/BbsI fragment
lacking the sequence encoding the V1V2 loop was introduced in place
of the corresponding fragment in the pCR2.1-TOPO-gp140.DELTA.V3 and
pCR2.1-TOPO-gp160.DELTA.V3 plasmids.
[0152] After BsiWI/BssHII digestion of the different pCR2.1-TOPO
plasmids, the native and mutant gp160 and gp140 sequences were
cloned in the EdB-tag vector in ATU position 2 and ATU position 3
(FIG. 2B). The resulting plasmids were designated
pMV2-gp160.sub.HIV, pMV2-gp140.sub.HIV.
[0153] Cells were maintained in Dubelbecco's modified Eagle's
medium (DMEM) supplemented with 5% fetal calf serum (FCS) for Vero
cells (African green monkey kidney), or with 10% FCS, 1 mg/ml G418
for helper 293-346 cells (35) and for P4-CCR5 cells
(Hela-CD4-CXCR4-CCR5-HIVLTR-LacZ) (12).
[0154] Recovery of Recombinant MV.sub.EdB-Env.sub.HIV89.6
virus.
[0155] To recover the recombinant MV.sub.EdB-HIV viruses from the
plasmids, the different EdB-HIV Env plasmids were used to transfect
293-346 helper cells.
[0156] To recover the measles virus from the EdB-HIV-Envplasmids
cDNA, we used the helper-cell-based rescue system described by
Radecke et al. (Radecke, F., et al 1995. Rescue of measles viruses
from cloned DNA. EMBO Journal. 14:5773-5784) and modified by Parks
et al. (Parks, C. L., et al. 1999. Enhanced measles virus cDNA
rescue and gene expression after heat shock. J. Virol.
73:3560-3566). Human helper cells stably expressing T7 RNA
polymerase and measles N and P proteins (293-346 cells, disclosed
by Radecke et al) were co-transfected using the calcium phosphate
procedure with the EdB-HIV-Env plasmids (5 .mu.g) and a plasmid
expressing the MV polymerase L gene (pEMC-La, 20 ng, disclosed by
Radecke et al). The virus was rescued after cocultivation of
transfected 293-346 helper cells at 37.degree. C. with primate Vero
cells (african green monkey kidney). In this case, syncytia
appeared systematically in all transfections after 2 days of
coculture.
[0157] In a further experiment (FIGS. 3C-D), after overnight
incubation at 37.degree. C., the cells were heat shocked at
43.degree. C. for 3 hours in fresh medium (40). Heat-shocked cells
were incubated at 37.degree. C. for 2 days, then transferred onto a
70% confluent Vero cells layer (10 cm Petri dishes). Syncytia
appeared in Vero cells after 2-5 days of co-culture. Single
syncytia were harvested and transferred to Vero cells grown in 35
mm wells. The infected cells were expanded in 75 and 150 cm3
flasks. When syncytia reached 80-90% confluence, the cells were
scraped in a small volume of OptiMEM (Gibco BRL) and frozen and
thawed once. After centrifugation, the supernatant, which contained
virus, was stored at -80.degree. C.
[0158] Expression of HIV1 Glycoproteins by Recombinant MV.
[0159] The rescued recombinant viruses MV2-gp140, MV2-gp160,
MV3-gp140.DELTA.V3 and MV2-gp160.DELTA.V3 were propagated on Vero
cells and the expression of HIV Env glycoproteins was analyzed by
western blotting and immunofluorescence. Infection of Vero cells by
recombinant MV2 viruses (with transgene insertion in position 2)
showed a high expression of the HIV Env gp160 and gp140. The
cleaved recombinant Env protein (gp120) was also detected. The MV3
virus (with transgene insertion in position 3) expressed lower
levels of transgene, as expected due to the transcription gradient
observed in MV expression. Taken together, these results indicate
that HIV1 Env glycoprotein and .DELTA.V3 mutant are efficiently
expressed by the recombinant MVs.
[0160] Virus titration. The titers of recombinant MV were
determined by an endpoint limit dilution assay on Vero cells. 50%
tissue culture infectious dose (TCID.sub.50) were calculated using
the Karber method.
[0161] Growth Capacity of the MV.sub.EdB-Env.sub.HIV89.6
Recombinant Viruses.
[0162] To analyze the growth capacity of MV.sub.EdB-Env.sub.HIV89.6
viruses, Vero cells were infected at different MOI (0.01 and
0.0001), incubated at 37.degree. C., and collected at different
time points. Titers of cell-associated viruses were determined for
each sample using the TCID.sub.50 method on Vero cells. FIG. 4
shows that using MOI of 0.0001, the growth kinetics of
MV.sub.EdB-Env.sub.HIV89.6 viruses was delayed, as compared to
standard MV.sub.EdB-tag. However, using an MOI of 0.01 the
production of recombinant viruses was comparable to that of
standard virus, and peak titers of 10.sup.7 TCID.sub.50/ml or even
more were easily obtained.
[0163] In particular, monolayers of Vero cells (T-25 flasks) were
infected at an MOI of 0.05 with the recombinant viruses. When
syncytia reached 80-90% confluence, cells were lysed in 150 mM
NaCl, 50 mM Tris pH=8, 1% NP40, 0.5 mM PMSF and 0.2 mg/ml Pefabloc
(Interbiotech, France). Chromatin was removed by centrifugation and
the concentration of protein in the supernatant was determined with
a Bradford assay. Proteins (50 .mu.g) were fractionated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to cellulose membranes (Amersham Pharmacia Biotech).
The blots were probed with a mouse monoclonal anti-HIV gp120
antibody (Chessie 13-39.1, NIH-AIDS Research & Reference
Reagent Program) or with a monoclonal anti-MV N antibody (Chemicon,
Temecula, USA). A goat anti-mouse IgG antibody-horseradish
peroxidase (HRP) conjugate (Amersham) was used as second antibody.
Peroxidase activity was visualized with an enhanced
chemiluminescence detection Kit (Pierce).
[0164] Mice Immunizations
[0165] Mice susceptible for MV infection were obtained as described
previously (21). Transgenic FVB mice heterozygous for CD46 (32),
the receptor for MV vaccine strains (24) were crossed with 129sv
IFN-.alpha./.beta.R.sup.-/- mice lacking the type I interferon
receptor (22). The F1 progeny was screened by PCR and the
CD46.sup.+/- animals were crossed again with 129sv
IFN-.alpha./.beta.R.sup.-/- mice. IFN-.alpha./.beta.R.sup.-/-
CD46.sup.+/- animals were selected and used for immunization
experiments. The same type of mice have already been shown to be
susceptible to MV infection (20, 21).
[0166] Six-weeks-old female CD46.sup.+/-
IFN-.alpha./.beta.R.sup.-/- mice were inoculated intraperitoneally
with 10.sup.7 TCID.sub.50 of MV2-gp140, MV2-gp160,
MV3-gp140.DELTA.V3 or MV2-gp160.DELTA.V3 recombinant viruses
prepared and titrated as described above. Mice were euthanized 7
days and 1 month post-infection. Spleens and whole blood were
collected. Splenocytes were extracted from spleens and kept frozen
in liquid nitrogen until use. Serums were decanted and serology was
analyzed by ELISA for MV (Trinity Biotech, USA) and HIV (Sanofi
Diagnostics, France).
Monkey Immunization
[0167] Two colony-bred rhesus macaques (Macaca mulatto)
(seronegative for simiam type D retrovirus, simian T-cell
lymphotropic virus, simian immunodeficiency virus and MV) were
vaccinated subcutaneously with 104 TCID50 of MV vaccine (Rouvax,
Aventis Pasteur, France). They were boosted one year later by two
injections of 5 106 TCID50 of MV2-gp140 recombinant virus done at 1
month interval. Blood samples were collected at different time
points and anti-MV and anti-HIV antibodies were looked for. Humoral
immune response to rescued recombinant viruses.
[0168] 1.sup.st Experiment
[0169] Humoral immune responses against MV and HIV Env were
analyzed by ELISA in serums collected 1 month after immunization of
mice. Titers were determined by limiting dilutions. The results
presented in FIG. 5 show that all the vaccinated mice responded to
measles with high titers of antibodies (1/50000 to 1/80000) and to
HIV Env with titers between 1/1000 and 1/5000 depending on the
inserted sequence. The antibody titers between MV and HIV cannot be
compared because the ELISA used have not the same sensitivity. The
MV ELISA (Trinity Biotech, USA) detected the whole response against
all MV proteins, while the HIV ELISA (Sanofi Diagnostics) detected
only the anti-HIV Env antibodies. The capacity of these sera to
neutralize a primary HIV lade B isolate was tested using indicator
cells, P4R5, that express beta-galactosidase when infected with HIV
(HeLa-CD4-CXCR4-CCR5-HIV LTR-LacZ cells). In preliminary
experiments, we tested sera of mice immunized with recombinant
MV-HIV viruses expressing native envelope glycoproteins
(MV-gp.sup.160.sub.HIV-1 or MV.sub.EdB-gp.sup.140.sub.HIV89.6). The
results showed that these sera had a 70-50% neutralizing activity
against a primary isolate, Bx08, when used at a 1/20 dilution (FIG.
6). The neutralizing activity of sera raised against the
genetically engineered Env molecules is currently under study.
[0170] 2.sup.nd Experiment
[0171] In another experiment (FIG. 5C-F), sera were collected one
month after immunization and heat inactivated. Anti-MV (Trinity
Biotech, USA) and anti-HIV Env (Sanofi Diagnostic Pasteur, Biorad,
France) antibodies were deteced using commercial ELISA kits. An
anti-mouse antibody-HRP conjugate (Amersham) was used as the
secondary antibody. Titers were determined by limiting dilutions
and calculated as the highest dilution of serum giving twice the
absorbence of a 1/100 dilution of a mixture of control sera. The
same ELISA kits were used for sera from macaque monkeys. An
anti-monkey IgG secondary antibody was used to detect anti-HIV
antibodies. Anti-MV antibodies were detected with an anti-humn IgG
in order to be able to calibrate the assay with standards supplied
in the MV ELISA kit. They were expressed in mIU/ml. A mixture of 5
samples from negative monkeys was used as the negative control. The
titer of anti-ELDKAS antibodies was determined by ELISA using
96-well NeutrAvidin plates (Pierce) coated with the ELDKWAS
biotynilated peptide (Neosystem, 5 .mu.g/ml in NaHCO.sub.3 2M,
Na.sub.2CO.sub.3.H.sub.2O 2M, pH 9.6). Sera from mice immunized
with standard MV were used as negative controls. Peptide-bound
antibodies were detected with anti-mouse antibody-HRP
conjugate.
[0172] HIV-1 neutralization assays. Sero-neutralization was tested
against SHIV89.6p (A. M. Aubertin, Universite Louis Pasteur,
Strasbourg, H. Fleury, Bordeaux, France), 92US660, 92US714, 92HT593
(NIH-AIDS Research & Reference Reagent Program), and a clade A
primary isolate: 3253 (G. Pancino, Institut Pasteur, Paris). These
viruses were propagated on PHA-stimulated human PBMC as already
described (42). HIV-1 neutralization assays were performed using
the P4-CCR5 indicator cell line (43). P4-CCR5 cells were seeded in
96-well plates (20 000 cells per well) and incubated at 37.degree.
C. in DMEM, 10% FCS for 24 h. The medium was replaced by 100 .mu.l
DMEM, 10% FCS, DEAE dextran (100 .mu.g/ml) and the cells were
incubated at 37.degree. C. for 30 minutes. Virus (0.5 ir 1 ng p24)
was incubated with serum dilutions in 50 .mu.l PBS at 37.degree. C.
for 20 minutes and the virus-serum mixtures were added to the cells
in triplicate. After 48 hours of incubation, the
.beta.-galactosidase activity was measured using a
Chemiluminescence Reporter Gene Assay (Roche, USA).
[0173] Cellular Immune Responses to Rescued Recombinant
Viruses.
[0174] The capacity of splenocytes from vaccinated mice to secrete
.alpha.-IFN upon in vitro stimulation was tested by flow-cytometry
and ELISpot assays. Frozen cells from immunized mice were thawed 18
h before functional assays and incubated in RPMI medium
supplemented with 10% 56.degree. C.-heated FCS (Gibco) and 10 U
rh-IL2 (Boehringer Mannheim). Cell viability was evaluated by
trypan-blue exclusion.
[0175] To perform .gamma.-IFN ELISpot assay, multiscreen-HA
96-wells plates were coated with capture anti-mouse .gamma.-IFN
(R4-6A2, Pharmingen) in PBS solution (6 .mu.g/ml). After overnight
incubation at 4.degree. C., wells were washed 4 times with PBS. The
remaining protein binding sites were blocked by incubating wells
with 100 .mu.l RPMI/FCS 10% for 1 h at 37.degree. C. Medium was
withdrawn just before addition of cell suspensions (100 .mu.l) and
stimulating agents (100 .mu.l). Splenocytes from immunized mice
were plated at 5.10.sup.5 cell per well in duplicate in RPMI.
Concanavalin A (5 .mu.g/ml, Sigma) was used as a positive control,
and RPMI/IL2 (10 U/ml) as a negative control. Cells were stimulated
either with 1 .mu.g/ml HIV1 gp120, 1 .mu.g/ml Bovine Serum Albumin
(Sigma), or Edm-Tag virus (MOI=1). After incubation for 2 h at
37.degree. C. for viral adsorption, heated-FCS (10 .mu.l) was added
in each well (10% final concentration) and plates were incubated
for 24-36 h at 37.degree. C. To remove cells, the plates were
washed twice with PBS, 4 times with PBS containing 0.05% Tween 20
(Sigma), and 2 times again with PBS. For detection, a biotinylated
anti-mouse .gamma.-IFN antibody (XMG1.2, Pharmingen) was added to
each well (100 .mu.l, 4 .mu.g/ml in PBS-0.1% FCS). After incubation
for 2 h at room temperature, plates were washed 4 times with
PBS-0.1% tween 20 and twice with PBS. Streptravidin-Alkaline
Phosphatase (AP) conjugate (Roche) (100 .mu.l, 1/2000 dilution in
PBS) was added and incubated for 1-2 hours at room temperature. The
enzyme was removed by 4 washes with PBS-0.1% Tween 20 and 2 washes
with PBS. Spots were then developed with BCIP/NBT color substrate
(Promega) prepared in AP buffer pH 9.5 (1 M Tris, 1.5 M NaCl, 0.05
M MgCl2). Wells were monitored for spot formation by eye: after a
15-30 minutes incubation the reaction was stopped by washing under
running tap water. After drying at least overnight at room
temperature, colored spots were counted using an automated image
analysis system ELISpot Reader (Bio-Sys).
[0176] For Flow-cytometry assays, 5 10.sup.5 splenocytes (diluted
in 100 .mu.l RPMI) were stimulated in V-bottomed 96-wells plates
with either 1 .mu.g/ml HIV1 gp120 protein (AbCys) in RPMI/IL2 (10
U/ml), or EdB-tag virus (MOI=1) diluted in 100 .mu.l RPMI/IL2. Non
stimulated control cells were incubated with RPMI/IL2 (10 U/ml).
After incubation for 2 h at 37.degree. C. for viral adsorption, 10
.mu.l FCS were added in each well (10% final concentration) and
plates were incubated overnight at 37.degree. C. The medium was
then replaced by 150 .mu.l RPMI-10% FCS containing 10 U rh-IL2 and
10 .mu.g/ml Brefeldin A (Sigma). Cells were incubated for 4 hours
at 37.degree. C., harvested, stained with anti-mouse CD8-APC
(Pharmingen) and anti-mouse CD4-CyCr (Pharmingen) for 20 minutes at
room temperature, washed with PBS-BSA (0.5%), then fixed for 5
minutes at 37.degree. C. in CytoFix (Pharmingen). After washing
cells were resuspended in 100 .mu.l PBS-BSA (0.5%) containing 0.1%
Saponin (Sigma) and incubated for 30 minutes at room temperature
with anti-mouse .gamma.-IFN-PE (Pharmingen). Cells were washed
again and samples were analyzed using a FACSCalibur cytometer
(Becton Dickinson). The data were analyzed using Cell Quest
software.
[0177] Recombinant MV Express HIV89.6 Env Glycoproteins and
Replicate Efficiently.
[0178] The anchored (gp160) and soluble (gp140) forms of the HIV
Env glycoprotein (strain SHIV89.6p), with or without deletion of
the V3 loop and insertion of an additional ELDKWAS epitope, were
inserted into one of the ATU of the p(+)MV vector (FIG. 2).
Recombinant viruses MV2-gp140, MV2-gp160, MV3-gp140.DELTA.V3 and
MV2-gp160.DELTA.V3 were obtained after transfection of the plasmids
into the 293-346 helper cell line and propagation in Vero cells.
MV2- and MV3- refers to the site of the insertion, position 2 or 3
respectively, of the EnvHIV89.6 construction. Expression of the
EnvHIV89.6 protein was analyzed by western blotting of
infected-cells lysates (FIG. 3) and immunofluorescence (not shown).
The MV2-gp140 and MV2-gp160 viruses showed a high level of
expression of the EnvHIV89.6 protein (FIG. 3C, lanes 1, 2, 4). As
expected, the MV2-gp160.DELTA. viruses expressed the env gp160
precursor as well as the cleaved gp120 protein (FIG. 3C, lanes 2,
4). In contrast, the MV2-gp140 and MV3-gp140.DELTA.V3 viruses
expressed only the secreted, uncleaved gp140 form. The
MV3-gp140.DELTA.V3 virus expressed slightly lower levels of
transgene than viruses of the MV2- series, as expected, due to the
transcription gradient observed in MV expression (FIG. 3C, lane 3).
Taken together, these results indicate that Env.sub.HIV89.6 and the
.DELTA.V3 mutants were efficiently expressed and correctly matured.
The recombinant MV were passaged 5 times on Vero cells and the
expression of the transgene was compared to that of the MV
nucleoprotein. FIG. 3 shows that Env.sub.HIV89.6 expression was
similar for passages 2 and 5, confirming the stability of
expression of transgenes in this system.
[0179] The growth of MV-Env.sub.HIV89.6 recombinant viruses was
analyzed on Vero cells using an MOI of 0.0001 or 0.01. The growth
of recombinant viruses was only slightly delayed compared to that
of standard EdB-tag MV rescued from p(+)(MV). Viruses expressing
the secreted gp140 were less affected than viruses expressing the
anchored gp160. The gp140.DELTA.V3 recombinant grew at the same
rate as control MV. The delay observed with viruses expressing the
anchored gp160 may be due either to lower replication rate, because
of the larger size of the transgene, or to reduced MV budding
because of the insertion of gp160 at the surface of the infected
cells. Nevertheless, the final yield of recombinant viruses was
comparable to that of control MV and peak titers of about 10.sup.6
to 10.sup.7TCID50/ml were obtained routinely.
[0180] Induction of Humoral Immune Response to Recombinant MV in
Susceptible Mice.
[0181] The immunogenicity of MV-Env.sub.HIV89.6 viruses was tested
in genetically modified mice expressing the human CD46 MV receptor
and lacking the Type I IFN receptor. Increasing doses of MV2-gp160
virus (103-107 TCID50) were tested in 5 groups of 3 mice.
Antibodies to MV and HIV Env were looked for by ELIA in sera
collected 1 month after immunization (FIG. 5C). Both anti-MV and
anti-HIV antibody titers increased when the dose of recombinant MV
increased. Since high anti-MV titers were obtained when animals
were inoculated with 10.sup.6 to 10.sup.7 TCID.sub.50, mice were
immunized with 5.106 TCID.sub.50 in all further experiments. At
this dose, anti-MV antibody titers were six fold higher than
anti-HIV titers. One should keep in mind that immunization was
against HIV Env only, whereas all MV proteins were expressed during
infection. To compare the immunogenicity of the different
Env.sub.HIV constructs, four groups of 6 mice were inoculated
intraperitoneally with various MV-Env.sub.HIV89.6 viruses (FIG. 5B,
5E). All mice responded to MV (mean anti-MV titer: 5 10.sup.4) and
to HIV Env (mean anti-HIV titer: 8 10.sup.3). No difference in
anti-MV or anti-HIV or anti-HIV titers was observed between the
four constructs tested. Interestingly, expression from the ATU 2 or
the ATU 3 position of the MV vector did not affect the antibody
response. Because the .DELTA.V3 constructions expressed an
additional ELDKWAS epitope, the antibody response against this gp41
epitope was examined separately using a specific ELISA assay (FIG.
5F). The results showed that the .DELTA.V3-ELDKWAS constructions
induced higher titers of anti-ELDKWAS antibodies. The titer of 1/50
000 corresponds to the dilution of an immune serum capable of
recognizing the antigen administered for the immunization, in ELISA
assay.
[0182] MV-Env.sub.HIV89.6 Viruses Induce Neutralizing Anti-HIV
Antibodies.
[0183] The capacity of these sera to neutralize either homologous
SHIV89.6p virus or various heterologous primary HIV-1 isolates was
tested using a single cycle virus infectivity assay on P4-CCR5
indicator cells (43). P4-CCR5 cells express the CD4, CXCR4 and CCR5
HIV-1 receptors and have been stably transfected with an HIV LTR
LacZ. Therefore, they are susceptible to HIV-1 isolates and express
.beta.-galactosidase upon infection. The sero-neutralization assay
was validated using a combination of anti-HIV immunoglobulin
(HIVIG) and monoclonal antibodies (2F5 and 2G12) previously shown
to synergistically neutralize primary HIV isolates (17). We also
used sera from infected patients that neutralize primary HIV
isolates (17). We also used sera from infected patients that
neutralize primary HIV primary isolates using a standard
neutralization assay on human PBMCs (42). The neutralizing activity
of a serum (Table 1) is expressed as the ratio of the reduction of
infection obtained with this serum over the reduction obtained with
negative control sera used at the same dilution (sera from HIV
negative individuals and from infected patients neutralized lade B
and A viruses equally well in this assay.
[0184] As shown in Table 1, antibodies induced in mice by the four
MV-Env.sub.HIV89.6 viruses neutralized the homologous SHIV89.6p at
both dilutions tested (1/30 and 1/60). No significant difference
was observed between the sera obtained with the different Env
constructs, indicating that the secreted and anchored from of HIV
glycoprotein induced neutralizing antibodies against homologous
virus equally well when expressed by MV. Deleting the V3 loop,
known to contain type-specific neutralizing epitopes, had no
significant effect on the induction of antibodies that neutralized
the homologous virus. This suggests that the deletion might have
been compensated either by the addition of a second ELDKWAS gp41
neutralizing epitope, or by the uncovering of other neutralizing
epitopes.
[0185] The antibodies induced by the recombinant viruses
neutralized heterologous primary lade B isolates, except the
92HT593 isolate, as well as a lade A virus. In each case,
antibodies induced by the anchored gp160 were slightly more
neutralizing than antibodies induced by the secreted gp140,
especially against the lade A 3253 virus. The antibodies induced by
the .DELTA.V3-ELDKWAS Env.sub.HIV89.6 neutralized heterologous
viruses more efficiently than those induced by the native envelope.
This was particularly striking for the Bx08 virus which could be
neutralized up to 90% by sera from mice immunized with
MV2-gp160.DELTA.V3 (1/30 dilution) but not by sera from mice
immunized with MV expressing the native Env.sub.HIV89.6. This
neutralization was just as efficient as neutralization by positive
control sera. These results show that replacing the V3 loop of
Env.sub.HIV89.6 by an additional ELDKWAS gp41 epitope and
expressing the construct with a MV vector allowed the induction of
antibodies with cross-neutralizing activity against clade A and B
HIV-1 primary isolates, at least in the context of recombinant MV
infection of mice. TABLE-US-00009 TABLE 1 Neutralization of HIV-1
primary heterologous isolates by sera from MV- Env.sub.HIV89.6
immunized mice.sup.a. Positive controls Mice Sera (1/60) Mice Sera
(1/30) Human HIV MV2 MV2 MV2 MV2 Mab sera.sup.c Virus isolate MV2
Gp140 MV2 Gp160 MV2 Gp140 MV2 Gp160 (2F5/2G12/ 4 33 (subtype) Gp140
.DELTA.V3 Gp160 .DELTA.V3 Gp140 .DELTA.V3 Gp160 .DELTA.V3 HIV-IG
61/40) -1/30) SHIV 89.6 40 50 52 45 76 57 72 68 ND ND ND Bx08 (B) 0
31 0 40 0 76 18 90 94 94 90 92 US 660 (B) 2.5 15 13 17 ND ND ND ND
ND ND ND 92 US 714 (B) 45 49 45 68 ND ND ND ND ND ND ND 92 HT 593
(B) 0 0 0 0 0 0 0 0 ND ND ND 3253 (A) 0 0 18 30 0 10 43 49 73 54 45
.sup.aSerum was evaluated for neutralizing antibodies at two
dilutions. Values are % reduction in infection of primary HIV
isolates on P4-CCR5 cells in presence of mice sera (three mice per
point). Determinations were made in triplicate and the standard
deviations were <10%. .sup.bMix of HIVIG (2.5 mg/ml) and Mabs
2F5 and 2G12 (25 .mu.g/ml). .sup.cNumbers correspond to the
nomemclature used in Burrer et al.
[0186] Induction of Cellular Immune Response Against Recombinant
MV
[0187] The results of these experiments performed with splenocytes
from mice immunized with MV2-gp160.sub.HIV virus (FIG. 7)
demonstrated that a single immunization with MV2-gp160.sub.HIV
virus was able to prime HIV Env-specific lymphocytes in vivo. The
.gamma.-IFN-ELISpot assay is a sensitive method for
antigen-specific cell numeration in fresh cells after in vivo
immunization. This assay was used to determine whether
HIV-Env-specific .gamma.-IFN-secreting cells could be detected
after a single immunization with the MV2-gp160.sub.HIV virus. FIG.
7A shows that a significant number of Env-specific cells were
present in 2/3 mice tested, 7 days as well as 1 month after
immunization. (For one mouse in each group the number of spots was
the same after BSA or gp120 stimulation). The number of
HIV-specific spots detected (up to 600/10.sup.6 cells) represents
15-20% of MV-specific spots detected in the same mice (not shown),
indicating that recombinant MV is able to efficiently immunize
against the foreign gene expressed.
[0188] To assess the phenotype of these Env-specific cells, 3-color
cytofluorometry experiments were performed on mice euthanized 7
days after immunization, at the theoretical peak of effector cells
proliferation. A representative result is shown on FIG. 7B. The
background .gamma.-IFN production level for both CD4+ and CD8+
lymphocytes is shown on the left panel. For this animal, 0.09% of
CD8+ lymphocytes (mean calculated for 3 mice: 0.31%) and 0.25% of
CD4+ lymphocytes (mean: 0.41%) were spontaneously producing
.gamma.-IFN. The frequencies of HIV-gp120 T-cells (middle panel) in
the CD8+ and CD4+ subsets were 1.760% (mean: 1.69%) and 0.92%
(mean: 0.76%) respectively. It's interesting to take into account
that in the same immunized mouse the frequencies of Measles
specific cells in CD8+ and CD4+ subsets were 7.63% (mean: 7.03%)
and 4.11% (mean: 3.50%) respectively. Indeed the recombinant
MV2-gp160.sub.HIV virus expresses 6 measles proteins plus one gp160
foreign protein. Thus, the frequencies of antigen-specific
lymphocytes followed the recombinant gene proportions. As a
conclusion, 3-color cytofluorometry performed 7 days after
MV2-gp160.sub.HIV virus vaccination showed that both CD8+ (FIG. 7B,
upper panel) and CD4+ (FIG. 7B, lower panel) lymphocytes specific
for HIV gp120 and measles virus were primed in vivo. Inducing an
anti-HIV response in animals with pre-existing anti-MV
immunity.
[0189] We first tested the possibility of boosting the anti-HIV
response by a second injection of recombinant MV. Mice immunized
with 5.10.sup.6 TCID.sub.50 of MV2-gp140 recombinant virus (3 mice
per group) were boosted with a second injection of the same
recombinant MV one month after the first injection. The mean
anti-MV and anti-HIV antibody titers at the time of boosting were 5
10.sup.4 and 8 10.sup.3 respectively. These titers increased to,
respectively 5 10.sup.5 and 5 10.sup.4 one month after boosting.
Thus, anti-MV and HIV responses can be boosted 10 times by
injecting the same dose of recombinant MV one month after the first
immunization.
[0190] We then tested the ability of recombinant MV to induce
anti-HIV antibodies in mice and monkeys in the presence of
pre-existing anti-MV immunity. Mice (3 mice per point) were first
immunized with 10.sup.5 TCID.sub.50 of EdB-tag MV (without an HIV
insert). High levels of anti-MV antibodies were induced (FIG. 7C).
The titer decreased slightly after 2 months and remained stable for
the following 9 months. Mice were then inoculated with 5 10.sup.6
TCID.sub.50 of MV2-gp140.sub.HIV89.6, and boosted with the same
dose one month later. The titer of anti-MV antibodies was increased
100 times and high titers of anti-HIV antibodies (5 10.sup.4) were
induced. These titers were similar to those obtained after
immunization of naive animals with two injections.
[0191] The same experiment was performed with rhesus macaques (FIG.
7D). Two macaques were immunized with a standard dose (10.sup.4
TCID.sub.50) of MV vaccine (Rouvax, Aventis Pasteur). As for mice,
high anti-MV antibody levels were induced and remained stable
during one year. Macaques were then inoculated with 5 10.sup.6
TCID.sub.50 of MV2-gp140.sub.HIV89.6 twice at one month interval.
Anti-MV titers increased 150 times after the first injection of
MV-HIV, while the second injection had no or little effect.
Anti-HIV antibodies were induced by the first MV2-gp140.sub.HIV89.6
injection despite the presence of pre-existing anti-MV immunity.
One month after the second MV2-gp140.sub.HIV89.6 injection, the
anti-HIV antibody level had increased about 10 times and had
reached titers similar to those obtained in mice. This level
remained stable for the following 5 months.
[0192] The main goal of the present work was to test the
immunogenicity of attenuated MV-Env.sub.HIV recombinant viruses. We
showed that such recombinants were genetically stable, expressed
the HIV Env protein at high levels, and induced high titers of
antibodies against both MV and the HIV Env constructs in transgenic
mice. The anti-HIV antibodies titers were approximately 15-20% of
those of the anti-MV antibodies. This corresponds roughly to the
ratio of HIV/MV proteins expressed by the recombinant viruses. HIV
Env constructions with a deleted V3 loop and an additional ELDKWAS
gp41 epitope induced twice as much anti-ELDKWAS antibodies as
native constructions, suggesting that the native conformation of
the additional peptide was conserved in spite of its ectopic
position. A high level of HIV-specific CD8+ and CD4+ cells was also
induced. As much as 1.5-2% of the total CD8+ T-cells and 0.9% of
the total CD4+ T-cells were HIV-specific.
[0193] However, the most important aspect of our results is that
these anti-HIV antibodies were neutralizing for the homologous
SHIV89.6p virus as well as for several heterologous lade A and lade
B HIV-1 primary isolates. Interestingly, the anchored gp160
.DELTA.V3-ELDKWAS construction induced antibodies that neutralized
heterologous viruses more efficiently than those induced by the
native envelope. Their neutralizing titers were similar to those of
reference human HIV-neutralizing sera. The broader neutralizing
capacity of these antibodies could be due either to the addition of
a second ELDKWAS gp41 neutralizing epitope, or to the exposure of
previously masked conserved neutralizing epitopes. Several groups
have inserted the ELDKWAS epitope into various immunogenic
molecules (44, 45, 46, 47). These studies showed that the
conformational context in which the epitope is displayed is
essential for the induction of neutralizing antibodies. A
.beta.-turn-like constraint was shown to be the most likely
conformation structure of the ELDKWAS epitope recognized by the 2F5
neutralizing antibody (46). In our constructions, the insertion of
the short AAELDKWASAA epitope in place of the V3 loop, which is
flanked by .beta.-strands (28, 29), may have such a
.beta.-turn-like conformation.
[0194] It has been shown, already, that deleting the hyper-variable
loops of HIV Env can enhance its immunogenicity (3, 48, 39).
However, in previous studies neutralizing antibodies wre obtained
only after multiple injections of high amounts of soluble protein
(23), or with a "prime boost" regimen using very large amounts of
DNA and pure protein (3, 39). In contrast, we observed the same
levels of neutralizing antibodies in mice injected with a single
dose of MV-gp160.DELTA.V3-ELDKWAS. Good immunogenicity in our
system results probably from the fact that the HIV Env is expressed
and processed by the immune system n the same way as proteins from
the live MV vaccine, a highly potent immunogen. One may hope that
such levels of neutralizing antibodies could at least induce
partial protection in vaccinated individuals. Accordng to the data
of others (3, 39), it might be possible to increase the
immunogenicity of M-HIV Env recombinants even furter by deleting
the V1 and V2 loops of HIV gp120, notably to induce antibodies
directed against the CD4-binding site. However, it has been
recently reported that this receptor-binding site can escape from
the immune response by conformational and entropic masking
(49).
[0195] The presence of anti-MV immunity in nearly the entire adult
human population would seem to restrict the use of MV recombinants
to infants, an already worthy goal in any event. However, several
studies showed that revaccinating already immunized individuals
results in a boost of anti-MV antibodies, suggesting that the
attenuated live vaccine replicated and expressed its proteins in
spite of preexisting immunity (50). Under such circumstances, one
might hope to be able to vaccinate adults against a foreign antigen
with a MV recombinant. Indeed, our results demonstrate, both with
mice and macaques, that high levels of anti-HIV neutralizing
antibodies can be obtained in the presence of pre-existing anti-MV
immunity.
[0196] Various "prime-boost" regimen, using combinations of naked
DNA and viral vectors such a sMVA (1) or Adenovirus (29), gave
reasonable protection against a challenge with pathogenic
SHIV89.6p. In the present study, we show that a single injection of
MV is able to combine humoral and cellular responses at levels
similar to those induced by these comlex combinations.
[0197] The same recombinants have been prepared using the cloned
Schwarz strain as a vector. This should raise their immunogenicity
even further.
EXAMPLE II
Construction of Schwarz Measles Viruses (MVSchw) Expressing HIV-1
Antigens
[0198] In order to test their capacity as vaccine candidates
against HIV infection, we constructed several recombinant Schwarz
measles viruses (MV) expressing HIV-1 antigens. Different HIV-1
genes from different open reading frames were constructed and
introduced in additional transcription units in the Schwarz MV cDNA
that we previously cloned (pTM-MVSchw). After rescue of the
different recombinant Schwarz measles viruses, the expression of
the different HIV-1 proteins was analyzed by western blotting of
infected-cells lysates (FIGS. 3A-D).
[0199] Different immunogens were constructed from HIV-1 Env
glycoprotein (hereafter 1-8), Gag protein (hereafter 9), and Tat
protein (hereafter 10): [0200] 1. Secreted glycoprotein gp140 from
HIV-1 89.6p [0201] 2. Anchored glycoprotein gp160 from HIV-1 89.6p
[0202] 3. Secreted glycoprotein gp140 from HIV-1 89.6p deleted from
hypervariable region V3 and additional AAELDKWASAA epitope
(gp140.sub.HIV89.6 .DELTA.V3-ELDKWAS) [0203] 4. Anchored
glycoprotein gp160 from HIV-1 89.6p deleted from hypervariable
region V3 with an additional AAELDKWASAA epitope (gp160.sub.HIV89.6
.DELTA.V3-ELDKWAS) [0204] 5. Secreted glycoprotein gp140 from HIV-1
89.6p deleted from hypervariable regions V1-V2
(gp140.sub.HIV89.6.DELTA.V1V2) [0205] 6. Anchored glycoprotein
gp160 from HIV-1 89.6p deleted from hypervariable regions V1-V2
(gp160.sub.HIV89.6.DELTA.V1V2) [0206] 7. Secreted glycoprotein
gp140 from HIV-1 89.6p deleted from hypervariable regions V1-V2-V3
(gp140.sub.HIV89.6 .DELTA.V1 V2V3) [0207] 8. Anchored glycoprotein
gp160 from HIV-1 89.6p deleted from hypervariable regions V1-V2-V3
(gp160.sub.HIV89.6 .DELTA.V1V2V3) [0208] 9. Gag polyprotein
(p17p24, delta myr) from HIV-1 (clade B consensus) truncated from
the nucleoprotein ORF in. C-terminal
(p17p24.differential.myrHIV-1B) [0209] 10. Tat protein from HIV-1
89.6p (TatHIV.sub.89.6)
[0210] The HIV env genes encoding the different forms of the Env
protein were generated by PCR amplification from plasmid pSHIV-KB9
(NIH-AIDS Research & Reference Reagent Program). The specific
sequences were amplified using Pfu Turbo DNA polymerase
(Stratagene) and specific primers. To generate the different
deletions, overlapping fragments flanking the sequences to be
deleted were generated and annealed together by PCR. They were then
introduced by enzyme restriction cloning in place of the
corresponding fragment in the gp160 and gp140 sequences already
cloned in pCR2.1-TOPO plasmids (FIG. 1A). The different sequences
generated include a start and a stop codon at both ends and respect
the "rule of six", stipulating that the nucleotides number of MV
genome must be divisible by 6 (7, 8). After BsiWI/BssHII digestion,
the different HIV sequences were introduced in the pTM-MVSchw
vector in ATU position 2 or 3 (FIG. 1B). The resulting plasmids
were designated: [0211] 1. pTM-MVSchw2-gp140.sub.HIV [0212] 2.
pTM-MVSchw2-gp160.sub.HIV [0213] 3.
pTM-MVSchw2-gp140.DELTA.V3.sub.HIV [0214] 4.
pTM-MVSchw2-gp160.DELTA.V3.sub.HIV [0215] 5.
pTM-MVSchw2-gp140.sub.HIV .DELTA.V1V2 [0216] 6.
pTM-MVSchw2-gp160.sub.HIV .DELTA.V1V2 [0217] 7.
pTM-MVSchw2-gp140.sub.HIV .DELTA.V1V2V3 [0218] 8.
pTM-MVSchw2-gp160.sub.HIV .DELTA.V1V2V3 [0219] 9.
pTM-MVSchw2-Gag.sub.HIV (p17-p24 .DELTA.myr) [0220] 10.
pTM-MVSchw3-Tat.sub.HIV
[0221] A recombinant virus expressing both Gag and gp140 in both
positions 1 and 2 of the measles Schwarz vector was produced.
[0222] 11. pTM-MVSchw2-Gag.sub.SIV239 (p17-p24
.DELTA.myr)-3-gp140.sub.HIV
[0223] This virus expressed both proteins (Fig z). Such constructs
allow the production of HIV, SHIV or SIV assembled Gag-Env "virus
like particles" in cells infected by recombinant measles virus.
[0224] The HIV-1 immunogenic sequences represented in FIG. 16 have
been generated:
EXAMPLE III
Recombinant Measles Viruses Expressing Different Viral
Transgenes
[0225] In order to demonstrate the immunizing and protective
capacities of MV as a pediatric vaccination vector, a series of
recombinant measles viruses expressing different viral transgenes
(listed below) from other viruses were constructed and studied. The
results presented here were obtained with the old EdB-tag vector.
However, we have shown that the EdB-tag was 100 times less
immunogenic than the Schwarz vaccine. Thus MV.sub.EdB recombinant
viruses were inoculated at higher doses. All the inserted sequences
with good immunological records can be obviously inserted in the
Schwarz vector.
[0226] Viral genes which have been already inserted in the
recombinant measles viruses: TABLE-US-00010 HIV clade B 89.6P gp160
gp140 gp160.DELTA.V3 gp140.DELTA.V3 gp160.DELTA.V1V2
gp140.DELTA.V1V2 gp160.DELTA.V1V2V3 gp140.DELTA.V1V2V3 tat HIV
clade B consensus codon optimized Gag (p17-p24) SIV Mac 239 Nef
Nef.DELTA.Myr Nef29-236 Tat HTLV-I Env Gag (p19-p24) Tax
EXAMPLE IV
Recombinant Measles Viruses Expressing Env and NS1 from Yellow
Fever Virus have Immune Capacity
[0227] Because a pediatric bivalent vaccine against measles and
yellow fever should be useful, we constructed recombinant MV
expressing the Env and NS1 proteins from Yellow Fever Virus (YFV
17D204, Pasteur vaccine strain) and tested their capacity to
protect mice from a lethal YFV challenge.
[0228] Construction of MV-YFV Recombinant Plasmids.
[0229] The env gene was PCR amplified with Pfu polymerase using
primers that contain unique BsiWI and BssHII sites for subsequent
cloning in MV vector MV-YFVEnv5
(5'-TATCGTACGATGCGAGTCGTGATTGCCCTACTG-3') and MV-YFVEnv3
(5'-ATAGCGCGCTTATGTGTTGATGCCMCCCA-3'). The Env protein thus
generated (amino acids 270-753 in YFV polyprotein) contained the
signal peptide in N-terminal and a part of the tramsmenbrane region
in C-terminal. The NS1 sequence was PCR amplified in the same way
with Pfu polymerase using primers: MVYFVNS5 (5'-TATCGTACGATGAGAAACA
TGACMTGTCC-3') and MVYFVNS3 (5'-ATAGCGCGCTTAATGGCTTTCATGCGTTT
TCC-3'). The NS1 protein (amino acids 754-1122 in YFV polyprotein)
contained its signal peptide sequence. A start and a stop codon
were added at both ends of the genes as well as several nucleotides
after the stop codon in order to respect the "rule of six",
stipulating that the nucleotides number of MV genome must be a
multiple of 6 (7). Both env and NS1 fragments were cloned in
pCR2.1-TOPO plasmid (Invitrogen) and sequenced to check that no
mutations had been introduced. After BsiWI/BssHII digestion of the
pCR2.1-TOPO plasmids, the env and NS1 sequences were cloned in the
EdB-tag vector in ATU position 2 giving plasmids: EdB-Env.sub.YFV
and EdB-NS1.sub.YFV.
[0230] Recovery of Recombinant EdB-Env.sub.YFV and EdB-NS1.sub.YFV
Viruses.
[0231] EdB-Env.sub.YFV and EdB-NS1.sub.YFV plasmids were used to
transfect 293-3-46 helper cells as described above, and recombinant
viruses were rescued from transfected cells cocultivated with Vero
cells. Recombinant viruses were passaged two times on Vero cells
and tested for transgene expression.
[0232] Expression of YFV Proteins by Recombinant MV.
[0233] The rescued recombinant viruses MV2-Env.sub.YFV and
MV2-NS1.sub.YFV were propagated on Vero cells and the expression of
YFV proteins was analyzed by immunofluorescence. FIG. 9 shows that
syncytia of Vero cells infected by recombinant MV2-YFV viruses
showed a high expression of the YFV Env and NS1 proteins as
detected with a mouse anti-YFV polyclonal serum. In order to
determine whether the expression of YFV genes was stable, the
rescued recombinant viruses were serially passaged on Vero cells.
After 10 passages all the syncytia observed in infected cells were
positive for YFV (not shown). Taken together, these results
indicate that Env and NS1 proteins from YFV are efficiently and
stably expressed over several passages by the recombinant MVs.
[0234] Mice Immunization with MV-YFV Recombinant Viruses.
[0235] A mixture of both MV2-Env.sub.YFV and MV2-NS1.sub.YFV
viruses (10.sup.7 TCID.sub.50) was inoculated intraperitoneally to
six CD46.sup.+/- IFN-.quadrature./.quadrature.R.sup.-/- mice as
described above (see MV-HIV gp experiments). As a control, six
other mice received the same dose of standard measles vaccine.
After one month, mice were intracranially challenged with YFV
17D204 (10 LD.sub.50 determined on FVB mice). FIG. 10 shows that
65% of MV-YFV immunized animals were fully protected against the
challenge, while all animals vaccinated with standard MV died
between 6 and 7 days post-challenge. Moreover, a 4-days delay in
mortality was observed in mice immunized with MV-YFV, and these
mice did not die with the same encephalitic clinical symptoms than
mice vaccinated with standard MV vaccine. The disease was
attenuated and consisted of limb paralysis. It has to be noticed
that IFN-.quadrature./.quadrature.R.sup.-/- mice are much more
sensitive to viral infections than immunocompetent mice
(10.sup.2-10.sup.4 times). For this reason, the lethal dose
determined on immunocompetent mice was probably too high for
IFN-.quadrature./.quadrature.R.sup.-/- mice. The same experiment is
underway using several decreasing doses of YFV challenge
viruses.
[0236] In conclusion, this preliminary experiment shows that the
immune responses induced by recombinant MV against YFV proteins are
able to protect mice against a lethal challenge.
[0237] The above constructs were made by using the sequences
disclosed on FIGS. 12A and 12B.
[0238] The same principles for the preparation of constructs would
apply with sequences disclosed on FIGS. 12C and 12D.
EXAMPLE V
Vaccination Against WNV with a Live Attenuated Measles Virus
(Schwarz Strain) Expressing the Secreted form of the E Glycoprotein
of the WNV (West Nile Virus)
[0239] We constructed a recombinant Schwarz measles attenuated
virus expressing the WNV E soluble form and tested its capacity as
vaccine candidate against. WN encephalitis. The WN cDNA
corresponding to the sE protein of IS-98-ST1 strain of WNV was
introduced in an additional transcription unit in the Schwarz MV
cDNA (pTM-MVSchw CNCM I-2889). After rescue of the recombinant
Schwarz measles virus, its capacity to protect mice from a lethal
WNV encephalitis following intraperitoneal challenge was
tested.
[0240] A) Materials and Methods
[0241] A.1 Cells and WN Virus
[0242] The IS-98-ST1 strain of WN virus was produced on Aedes AP61
mosquito cells according to the protocol described in Despres et al
(51), Mashimo et al (52) and Lucas et al (53). The Vero-NK cell
clone used in this study was selected for its capacity to fuse
after infection with measles virus and to amplify the WN virus.
[0243] A.2 Titration of WN Virus on AP61 Mosquito Cells by
Immunodetection of Focuses Viral Replication (Focus Immuno Assay,
FIA).
[0244] The titration was performed according to the protocol
described in Despres et al (51), Mashimo et al (52) and Lucas et al
(53).
[0245] The infectious titer of WN virus on AP61 cells was
determined as focus forming units on AP61 cells (AP61 UFF/ml).
[0246] A.3 Purification of WN Virus Produced on AP 61 Cells.
[0247] The purification was carried out according to the protocol
described in Despres et al (51), Mashimo et al (52) and Lucas et al
(53).
[0248] Briefly, the viral particles present in supernatants of AP61
cells infected during 3 days with WN virus strain IS-98-ST1 (MOI
0.4) were concentrated in 7% PEG 6000 and then purified in 30-60%
discontinuous saccharose gradient and in 10-50% linear saccharose
gradient. WN virious in 30% saccharose were stored at -80.degree.
C. The obtained infectious titers were about 10.sup.10 AP61
FFU/ml.
[0249] A.4 Anti-WN Antibody Detection in ELISA
[0250] The anti-WN antibody titers of diluted sera (1:100) were
determined by ELISA on a given quantity of 10.sup.6 AP61 FFU of WN
IS-98-ST1 virions purified in saccharose gradient. The protocol is
described in Despres et al (1993) and Mashimo et al (2002).
[0251] A.5 Anti-WN Immune Sera
[0252] Anti-WN immune sera were collected in adult mice genetically
resistant to viral encephalitis (Mashimo et al--2002) which were
tested during at least one month with intraperitoneal inoculation
of 103 AP61 FFU of WN virus strain IS-98-ST1.
[0253] The anti-WN antibody titer of 1:100 diluted immunsera were
measured in ELISA and were about 1.4 DO units. The neutralizing
titers (TNRF90) of anti-WN sera were about 1600.
[0254] Ascites of mice (HMAF) against WN strain IS-98-ST1 were
obtained from animals which had been hyperimmunized with brain
homogenates of baby mice inoculated with WN. The ELISA titers of
anti-WN HMAF, diluted to 1:1000 were about 1 DO unit.
[0255] The anti-WN immune sera were used for indirect
immunofluorescence and for passive seroprotection assays against
the disease. Anti-WN HMAF were used for membrane immunodetection of
viral proteins.
A6. Construction of Recombinant Schwarz Measles Virus Expressing WN
sE
[0256] The WNV env gene encoding the secreted form of the protein
was generated by RT-PCR amplification of viral RNA purified from
viral particles (WNV IS-98-ST1 strain). The specific sequence was
amplified using Pfu Turbo DNA polymerase (Stratagene) and specific
primers that contain unique sites for subsequent cloning in
pTM-MVSchw vector: MV-WNEnv5
5'-TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3' (BsiWI site underlined) and
MV-WNEnv3 5'-ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3' (BssHII site
underlined). A start and a stop codon were added at both ends of
the gene. The whole sequence generated is 1380 nucleotides long,
including the start and the stop codons and respects the "rule of
six", stipulating that the nucleotides number of MV genome must be
divisible by 6 [Calain, 1993 (7); Schneider, 1997 (28)]. The Env
protein thus generated contains its signal peptide in N-term (18
aa) and no transmembrane region. Thus, It represents amino acids
275-732 in WNV polyprotein and has the following sequence:
TABLE-US-00011
.diamond-solid.atgagagttgtgtttgtcgtgctattgcttttggtggccccagcttac
agcttcaactgccttggaatgagcaacagagacttcttggaaggagtg
tctggagcaacatgggtggatttggactcgaaggcgacagctgcgtga
ctatcatgtctaaggacaagcctaccatcgatgtgaagatgatgaata
tggaggcggtcaacctggcagaggtccgcagttattgctatttggctc
cgtcagcgatctctccaccaaagctgcgtgcccgaccatgggagaagc
tcacaatgacaaacgtgctgacccagcttttgtgtgcagacaaggagt
ggtggacaggggctggggcaacggctgcggattatttggcaaaggaag
cattgacacatgcgccaaatttgcctgctctaccaaggcaataggaag
aaccatcttgaaagagaatatcaagtacgaagtggccatttttgtcca
tggaccaactactgtggagtcgcacggaaactactccacacaggttgg
agccactcaggcagggagattcagcatcactcctgcggcgccttcata
cacactaaagcttggagaatatggagaggtgacagtggactgtgaacc
acggtcagggattgacaccaatgcatactacgtgatgactgttggaac
aaagacgttcttggtccatcgtgagtggttcatggacctcaacctccc
ttggagcagtgctggaagtactgtgtggaggaacagagagacgttaat
ggagtttgaggaaccacacgccacgaagcagtctgtgatagcattggg
ctcacaagagggagctctgcatcaagctttggctggagccattcctgt
ggaattttcaagcaacactgtcaagttgacgtcgggtcatttgaagtg
tagagtgaagatggaaaaattgcagttgaagggaacaacctatggcgt
ctgacaaaggctttcaagtttcttgtgactcccgcagacacaggtcac
ggcactgtggtgttggaattgcagtacactggcacggatggaccttgc
aaagttcctatctcgtcagtggcttcattgaacgacctaacgccagtg
ggcagattggtcactgtcaacccttttgtttcagtggccacggccaac
gctaaggtcctgattgaattggaaccaccctttggagactcatacata
gtggtgggcagaggagaacaacagatcaatcaccattggcacaagtct
ggaagcagcattggcaaagcctttacaaccaccctcaaaggagcgcag
agactagccgctctaggagacacagcttgggactttggatcagttgga
ggggtgttcacctcagttgggaaggctgtctaa
MRVVFVVLLLLVAPAYSFNCLGMSNRDFLEGVSGATWVDLVLEGDSCVT
IMSKDKPTIDVKMMNMEAVNLAEVRSYCYLATVSDLSTKAACPTMGEAH
NDKRADPAFVCRQGVVDRGWGNGCGLFGKGSIDTCAKFACSTKAIGRTI
LKENIKYEVAIFVHGPTTVESHGNYSTQVGATQAGRFSITPAAPSYTLKLG
EYGEVTVDCEPRSGIDTNAYYVMTVGTKTFLVHREWFMDLNLPWSSAGS
TVWRNRETLMEFEEPHATKQSVIALGSQEGALHQALAGAIPVEFSSNTVK
LTSGHLKCRVKMEKLQLKGTTYGVCSKAFKFLGTPADTGHGTVVLELQY
TGTDGPCKVPISSVASLNDLTPVGRLVTVNPFVSVATANAKVLIELEPPFG
DSYIVVGRGEQQINHHWHKSGSSIGKAFTTTLKGAQRLAALGDTAWDFG
SVGGVFTSVGKAV*
[0257] After agarose gel purification, the PCR fragment was cloned
in pCR2.1-TOPO plasmid (Invitrogen) and sequenced to check that no
mutations were introduced. After BsiWI/BssHII digestion of the
pCR2. 1-TOPO plasmid, the DNA fragment was cloned in the pTM-MVSchw
vector in ATU position 2 giving plasmid: pTM-MVSchw-sE.sub.WNV
according to FIG. 13.
A7. Production of Recombinant Measles Virus Expressing WN sE
[0258] To recover recombinant MV from plasmid, we used the
helper-cell-based rescue system described by Radecke et al.
[Radecke, 1995 (35)] and modified by Parks et al. [Parks, 1999
(40)]. Human helper cells stably expressing T7 RNA polymerase and
measles N and P proteins (293-346 cells, a kind gift from M A
Billeter, University of Zurich) were transfected using the calcium
phosphate procedure with pTM-MVSchw-sE.sub.WNV plasmid (5 .mu.g)
and a plasmid expressing the MV polymerase L gene (pEMC-La, 20 ng).
After overnight incubation at 37.degree. C., the transfection
medium was replaced by fresh medium and a heat shock was applied
(43.degree. C. for two hours) [Parks, 1999 (40)]. After two days of
incubation at 37.degree. C., transfected cells were transferred on
a CEF cells layer and incubated at 32.degree. C. in order to avoid
adaptation of the Schwarz vaccine that was originally selected on
CEF cells and is currently grown on these cells. Infectious virus
was recovered between 3 and 7 days following cocultivation. The
recombinant virus was also rescued by the same technique after
cocultivation of transfected 293-3-46 helper cells at 37.degree. C.
with Vero cells (african green monkey kidney, clone Vero-NK). In
order to increase the yield of rescue and because these recombinant
viruses were prepared to be used be used in mice experiments, we
used Vero cells as producing cells in place of the usual chick
embryo fibroblasts (CEF). Single syncytia were harvested and
transferred to Vero cells grown in 35 mm wells in Dulbebecco's
modified Eagle's medium (DMEM) supplemented with 5% fetal calf
serum (FCS). The infected cells were expanded in 75 and 150 cm3
flasks. When syncytia reached 80-90% confluence (usually 36-48
hours post infection), the cells were scraped in a small volume of
OptiMEM (Gibco BRL) and frozen and thawed once. After low-speed
centrifugation to pellet cellular debris, the supernatant, which
contained virus, was stored at -80.degree. C. We have shown that
two passages of the Schwarz virus on Vero cells did not change its
immunogenic capacities in macaques.
A8. Titration of Recombinant MV-WN Virus
[0259] The titers of recombinant MV were determined by an endpoint
limit dilution assay on Vero cells. 50% tissue culture infectious
dose (TCID.sub.50) were calculated using the Karber method [Karber,
1931 (41)].
A9. Immunofluorescence Detection of WNV sE Expressed in Vero Cells
Infected by MV-WN sE Recombinant Virus.
[0260] The expression of the WN sE protein in cells infected by
recombinant MV-WN sE was detected by immunofluorescence. Vero cells
were grown on polyornithine-coated coverslips and infected by MV-WN
sE at an MOI of 0.05. After two days of infection, coverslips were
washed twice in PBS and fixed for 15 minutes in paraformaldehyde
(4% in PBS). In some cases, cells were permeabilized by Triton X100
(0.1%, 5 min). After two PBS washes, coverslips were incubated for
15 minutes at room temperature in PBS with 2% goat serum, then
incubated for 1 hour at room temperature with mouse anti-WNV immune
sera or mouse anti-WNV HMAF (see A5) diluted in PBS with 2% goat
serum. After washing in PBS, cells were incubated for 45 minutes at
room temperature with R-phycoerythrin-conjugated goat anti-mouse
IgG (SBA, Birmingham). Following washing in PBS, coverslips were
mounted on slides with fluoromount (Southern Biotech Associates
inc., Birmingham, Ala.).
A10. Anti-MV Antibody Detection by ELISA
[0261] Anti-MV antibodies were detected using a standard ELISA kit
(Trinity Biotech, USA). An anti-mouse antibody-HRP conjugate
(Amersham) was used as the secondary antibody. Titers were
determined by limiting dilutions and calculated as the highest
dilution of serum giving twice the absorbence of a 1/100 dilution
of a mixture of control sera.
A.11 Neutralization Test by Reduction of Viral Replication Focuses
(TNRF90) on VERO Cells.
[0262] Successive dilutions of sera were prepared for testing in
DMEM Glutamax with 2% decomplemented FCS (Fetal Calf Serum) in
tubes of 0.5 ml.
[0263] For 0.1 ml of diluted serum in DMEM Glutamax with 2% FCS,
0.1 ml of DMEM Glutamax/2% FCS containing 100 AP61 UFF of WN virus
strain IS-98-ST1 was added.
[0264] Control cell: 0.2 ml of DMEM 0.2% FCS
[0265] Control virus: 0.2 ml of DMEM Glutamax/2% FCS containing 100
AP61UFF of WN virus strain IS-98-ST1.
[0266] 2 hours with mild rotation at 37.degree. C.
[0267] Plates with 12 cups with .about.150 000 VERO HK cells per
cup which are grown in monolayers for 24 hours in DMEM Glutamax 5%
FCS
[0268] 1 washing in DMEM of cell layers.
[0269] Add 0.2 ml of DMEM Glutamax/2% SVF
[0270] Add 0.2 ml of a mixture serum/WN virus on cell layers.
[0271] Incubate 2 hours at 37.degree. C. in CO.sub.2.
[0272] Withdraw the serum/WN virus mixture of infected cell
layers.
[0273] 1 washing in DMEM of infected cell layers.
[0274] Add 1 ml of DMEM 2% SVF per cup.
[0275] Add 1 ml of CMC 1.6% diluted in DMEM Glutamax/2% SVF
[0276] Incubate 2 days at 37.degree. C. in CO.sub.2.
[0277] The plaques were revealed through FIA technique. The last
dilution of immunsera which neutralize at least 90 of 100 UFF of WN
virus tested on VERO cells were determined (TNRF90: Test de
Neutralisation par Reduction de Foyers de replication virale a
90%). The titer of neutralizing antibodies of the sera was
determined by TNRF90.
A.12 Production of WN Virus Pseudo-Particles by Cell Line
MEF/3T3.Tet-Off/pr ME.WN #h2.
[0278] Pseudo-particles of WN virus strain IS-98-ST1 composed of
prME complexed glycoproteins were secreted by MEF/3T3.Tet-Off/pr
ME.WN #h2 line induced for the expression of viral proteins (CNCM
I-3018). They were purified for supernatants of 3-day cell culture
according to the protocol used for WN virus purification.
[0279] Passive seroprotection assay against WN virus in adult
BALB/c mice.
[0280] 6-week-old BALB/c mice were provided by the Janvier breeding
Center. The dose for viral test is 100 ap61 UFF, i.e. 10 DL 50
(Tomoshi et al 2002) diluted in 100 .mu.l of DPBS supplemented with
0.2% BSA (Bovine Serum Albumine) pH7.5 (Sigma) which are inoculated
intraperitoneally. The average time for lethal effect was 10 days.
Animals were observed for 2 to 3 weeks.
[0281] The sera to be tested for passive seroprotection in mice are
diluted in 0.1% DPBS/0.2% BSA and inoculated 24 hours prior to
viral test.
[0282] B) Results and Conclusions
B1. Production of Recombinant Measles Virus Expressing WN sE
[0283] cDNA encoding E protein of WNV strain IS-98-ST1 deleted for
its transmembrane anchoring region was inserted in the genome of
measles virus (Schwarz strain) according to FIG. 13.
B.2. Preliminary Assays of Passive Seroprotection Against WN Virus
in Mice
[0284] Anti-WN immune sera to be tested were obtained from mice
genetically resistant to the disease (52). The anti-WN sera, late
taken, were injected at dilutions 1:10 (16 TNRF90) et 1:40 (4
TNRF90) in a final volume of 0.1 ml DPBS/0.2% SAB intraperitoneally
in adult BALB/c mice genetically sensitive.
[0285] The antibodies were administered only 24 hours prior to the
viral test or 24 hours before and 24 hours after the test with 10
DL50 of strain IS-98-ST1 of WN virus. The negative control was the
injection of normal serum of mice at 1:10. The neurovirulence of WN
virus was evaluated in mice tested with DPBS/0.2% SAB. The results
of passive protection after two weeks of viral tests were as
follows: TABLE-US-00012 TABLE 1 Passive seroprotection against WNV
encephalitis in adult BALB/c mice. Passive transfer Mortality MDOD*
PBS/BSA (0.2%) 6\6 10.5 (.+-.1.5) normal serum (1:10) 6\6 12.5
(.+-.1.5) anti-WNV serum (1:10), 2 doses** 0\6 NA anti-WNV serum
(1:40), 2 doses 0\6 NA anti-WNV serum (1:10), 1 dose*** 1\6 12
anti-WNV serum (1:40), 1 dose 0\6 NA (*Mean Day Of Death .+-. SD)
(**Day-1 and Day+1 of virus challenge) (***Day-1 of virus
challenge)
[0286] To conclude, a unique injection of anti-WN antibodies (2.5 a
10 .mu.l of serum) obtained from mice genetically resistant to WN
virus, said injection being carried out intraperitoneally in adult
mice sensitive to viral encephalitis provides passive protection
against a test dose.
[0287] It is noted that the sera of BALB/c mice having received
anti-WN protective antibodies and resisting to viral infection have
anti-WN antibody titers by ELISA which are of about 1 DO unit (for
a dilution of serum of 1:100) after one month of test. This
indicates that the WN virus inoculated for the test has achieved
replication in protected mice, inducing a humoral response. If
passive seroprotection protects against lethal encephalitis due to
WN virus, it does not seem to be appropriate in order to prevent
viral propagration in infected individual.
B.3. Vaccination of CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/- mice
with MV/WN sE virus
[0288] Mice susceptible for MV infection were obtained as described
previously [Mrkic, 1998 (21)]. FVB mice heterozygous for the CD46
MV receptor transgene [Yannoutsos, 1996 (32)] were crossed with
129Sv IFN-.alpha./.beta.R.sup.-/- mice [Muller, 1994 (22)]. The F1
progeny was screened by PCR and the CD46.sup.+/- animals were
crossed again with 129Sv IFN-.alpha./.beta.R.sup.-/- mice.
IFN-.alpha./.beta.R.sup.-/- CD46.sup.+/- animals were selected and
used for immunization experiments. Six-week-old CD46.sup.+/-
IFN-.alpha./.beta.R.sup.-/- mice were inoculated intraperitoneally
with a single dose of standard MV vaccine (10.sup.6 TCID.sub.50. 3
mice) or MV-WN sE recombinant virus (10.sup.4 or 10.sup.6
TCID.sub.50, 6 mice per dose) in 300 .mu.l phosphate buffer saline
(PBS).
[0289] A serum has been taken from eye after one month of
vaccination with a unique dose in order to determine the production
of anti-MV, anti-WN E and neutralizing antibodies against the test
virus.
[0290] b) Sera Diluted to 1:100 and Tested for Antibodies by ELISA
on Purified NV Virion, for: TABLE-US-00013 DO unit Ascite of
anti-WN mice: 1 (control+) Serum of anti-WN mice: 0.8 (control+)
Serum of MV vaccinated mice: 0.110 .+-. 0.005 Serum of MV/WN sE
vaccinated mice, 10.sup.4 DCIP50: 0.635 .+-. 0.040 (males) Serum of
MV/WN sE vaccinated mice, 10.sup.4 DCIP50: 0.815 .+-. 0.005
(females) Serum of MV/WN sE vaccinated mice, 10.sup.6 DCIP50: 0.800
.+-. 0.200 (males) Serum of MV/WN sE vaccinated mice, 10.sup.6
DCIP50: 0.900 .+-. 0.195 (females)
c) In Vitro Seroneutralization Test for WNV on VERO Cells.
[0291] TNRF90 of pools of sera on 100 AP61UFF of strain IS-98-ST1
of WN virus in VERO cells: TABLE-US-00014 TNRF90 Serum of MV
vaccinated mice: <10 Serum of MV vaccinated mice MV-WN sE,
10.sup.4 DCIP50: 400 Serum of MV vaccinated mice MV-WN sE, 10.sup.6
DCIP50: 800
[0292] To conclude, antibodies directed against soluble E
glycoprotein WN virus have the capacity to neutralize strain
IS-98-ST1 used for the test by WN virus in mice in vitro.
[0293] A vaccine boost in immunized CD46.sup.+/-
IFN-.alpha./.beta..sup.-/- mice has been carried out 1 month after
the beginning of vaccination with a unique dose, identical to the
dose of the first injection.
[0294] After 2 weeks of boosting, sera were tested by ELISA and in
TNRF90 as above:
[0295] a) Sera Diluted to 1:100 and Tested for Antibodies by ELISA
on Purified WN Virion: TABLE-US-00015 DO Unit Ascite of anti-WN
mice: 1.4 (control+) Serum of anti-WN mice: 1 (control+) Serum of
MV vaccinated mice: 0.110 .+-. 0.005 Serum of MV-WN sE vaccinated
mice, 10.sup.4 DCIP50: 0.810 .+-. 0.100 (males) Serum of MV-WN sE
vaccinated mice, 10.sup.4 DCIP50: 1.150 .+-. 0.015 (females) Serum
of MV-WN sE vaccinated mice, 10.sup.6 DCIP50: 0.965 .+-. 0.230
(males) Serum of MV-WN sE vaccinated mice, 10.sup.6 DCIP50: 1.075
.+-. 0.240 (females)
b) Seroneutralization Test In Vitro on VERO Cells
[0296] TNRF90 of pools of sera on 100 AP61UFF of strain IS-98-ST1
of WN virus in VERO cells: TABLE-US-00016 TNRF90 Serum of boosted
MV mice: <10 Serum of boosted MV-WN sE, 10.sup.4 DCIP50 mice:
>1600 Serum of boosted MV-WN sE, 10.sup.6 DCIP50 mice:
>1600
[0297] After 4 weeks of boosting, the sera were tested by ELISA and
in TNRF90 as above:
[0298] a) Sera Diluted at 1:100 and Tested for Antibodies by ELISA
on Purified WN Virion: TABLE-US-00017 DO unit Ascite of anti-WN
mice: 1.7 (control+) Serum of anti-WN mice: 1.2 (control+) Serum of
MV vaccinated mice: 0.2 Serum of MV-WN sE vaccinated mice, 10.sup.4
DCIP50: 1.52 (.+-.0.15) Serum of MV-WN sE vaccinated mice, 10.sup.6
DCIP50: 1.76 (.+-.0.10)
b) Seroneutralization In Vitro on VERO Cells
[0299] TNRF90 of pools of sera on 100 AP61UFF of strain IS-98-ST1
of WN virus on VERO cells: TABLE-US-00018 TNRF90 Serum of MV-WN sE
vaccinated mice, 10.sup.4 DCIP50: 4000 (males) Serum of MV-WN sE
vaccinated mice, 10.sup.4 DCIP50: 8000 (females) Serum of MV-WN sE
vaccinated mice, 10.sup.6 DCIP50: 10 000-12 000
[0300] To conclude, after a boost with a unique dose, the anti-WNV
antibody titers and the anti-WNV neutralizing antibody titers were
significantly increased by a 10-fold factor or more.
[0301] Splenocytes of CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/- mice
immunized with two injections separated by 4 weeks with the MV-WN
sE virus with doses of 10.sup.4 or 10.sup.6 DCIP50 are tested in
ELISpot and flux/cytometry for the T CD4 and CD8 response after in
vitro stimulation with purified viral pseudo-particules in
saccharose gradients starting from supernatants of induced
MEF/3T3.Tet-Off/prME.WN #h-2 (CNCM I-3018) cell line.
B.4. Pasive Anti-WN Seroprotection Test in BALB/c with Anti-E
Antibodies
[0302] Immune sera of CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/- mice
vaccinated with a unique dose of recombinant measles virus has been
collected after one month. Various dilutions of these sera have
been injected in a final volume of 0.1 ml in 6-week-old BALB/c mice
and only 24 hours before inoculation of 100 AP61UFF of strain
IS-98-ST1 of WN virus (10 DL50) intraperitoneally (see protocol in
.sctn. B2).
[0303] The results of passive protection after two weeks of viral
test are as follows: TABLE-US-00019 TABLE 2 Recombinant MV-WN sE
induce antibodies that provide full protection against WNV
encephalitis in BALB/c mice Passive transfer Mortality Day PBS/BSA
(0.2%) 6\6 10 to 11 anti-WNV serum (1:10), 1 dose* 0\6 NA anti-WNV
serum (1:40), 1 dose 1\6 20 anti-MV (1:10), 1 dose 4\6 10 to 11
anti-MV-WN sE 10e4 (1:10), 1 dose 3\6 8 to 10 anti-MV-WN sE 10e6
(1:10), 1 dose 0\6 NA anti-MV-WN sE 10e6 (1:40), 1 dose 0\6 NA
anti-MV-WN sE 10e6 (1:100), 1 dose 3\6 10 to 11 (*Day-1 of virus
challenge)
[0304] To conclude, antibodies directed against WN-virus soluble
glycoprotein E have the capacity to protect in vivo against
WN-virus encephalitis. The vaccination of CD46.sup.+/-
IFN-.alpha./.beta.R.sup.-/- mice with a dose of 10.sup.6 DCIP50 of
MV-WN sE virus as a unique injection is required to induce an
anti-WN E humoral response on a four-week period of time which is
capable of protecting against the disease by passive
seroprotection. A minimal volume of 2.5 .mu.l of immune serum of
mice vaccinated with MV-WN sE virus, is sufficient to provide a
complete protection in adult BALB/c mice tested with a lethal dose
of WN-virus (i.e., a ratio of about 0.1 ml of immune serum/kg). It
is noted that anti-lethal sera diluted to 1:10 induce a partial
protection (about 30%) against West Nile virus encephalitis. Sera
obtained in vaccinated CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/-
mice which have then been boosted with a weak dose (10.sup.4
TCID50) will be tested for their capacity to provide passive
protection in BALB/c mice.
B.5. Viral Test on CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/- Mice
Vaccinated with MV-WN sE
[0305] CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/- mice vaccinated 2
months after the 2 injections of 10.sup.6 DCIP50 of MV-WN sE virus,
these injections being done at 4 weeks internal have been tested
with 100 AP61UFF of strain IS-98-ST1 of WN virus administered
intraperitoneally.
[0306] The 2 mice vaccinated with standard measles virus died the
3rd day of the test. No morbidity or lethality was observed for
mice vaccinated with MV-WN sE on the 7.sup.th day of the test. To
conclude, CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/- mice immunized
against soluble gpE of WN virus are protected against a lethal test
dose of WN virus in the absence of anti-viral activity of
alpha-interferon.
B6. New Test of Anti-WN Vaccination with an Antigen Boost
[0307] Adult CD46.sup.+/- IFN-.alpha./.beta.R.sup.-/- mice are
vaccinated on a 4 week period of time with MV-WN sE virus at a dose
of 10.sup.4 DCIP50 which is proposed for human and a boost with an
antigen is carried out with purified pseudo-particles of WN-virus
which are secreted by the cell line MEF/3T3.Tet-Off/WN prME #
h2.
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Sequence CWU 1
1
43 1 30 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 tatcgtacga tgagagtgaa ggagaaatat 30 2
30 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 2 atagcgcgca tcacaagaga gtgagctcaa 30 3
32 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 3 tatgcgcgct tatcttatat accacagcca gt 32
4 24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 4 ataagacatt caatggatca ggac 24 5 48 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 5 tgcccattta tccaattctg cagcattgtt gttgggtctt
gtacaatt 48 6 44 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 6 gataaatggg caagtgctgc
aagacaagca cattgtaaca ttgt 44 7 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 7
ctactcctat tggttcaatt ctta 24 8 11 PRT Human immunodeficiency virus
type 1 8 Ala Ala Glu Leu Asp Lys Trp Ala Ser Ala Ala 1 5 10 9 30
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 9 atttaaagta acacagagtg gggttaattt 30 10
42 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 10 gttactttaa attgtaacac ctcagtcatt
acacaggcct gt 42 11 30 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 11 ttgcataaaa
tgctctccct ggtcctatag 30 12 33 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 12 tatcgtacga
tgcgagtcgt gattgcccta ctg 33 13 30 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 13
atagcgcgct tatgtgttga tgccaaccca 30 14 30 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 14
tatcgtacga tgagaaacat gacaatgtcc 30 15 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 15
atagcgcgct taatggcttt catgcgtttt cc 32 16 18967 DNA Artificial
Sequence Description of Artificial Sequence Complete nucleotide
sequence of the pTM-MVSChw plasmid (CNCM I-2889) 16 gcggccgcta
atacgactca ctatagggcc aactttgttt ggtctgatga gtccgtgagg 60
acgaaacccg gagtcccggg tcaccaaaca aagttgggta aggatagttc aatcaatgat
120 catcttctag tgcacttagg attcaagatc ctattatcag ggacaagagc
aggattaggg 180 atatccgaga tggccacact tttaaggagc ttagcattgt
tcaaaagaaa caaggacaaa 240 ccacccatta catcaggatc cggtggagcc
atcagaggaa tcaaacacat tattatagta 300 ccaatccctg gagattcctc
aattaccact cgatccagac ttctggaccg gttggtgagg 360 ttaattggaa
acccggatgt gagcgggccc aaactaacag gggcactaat aggtatatta 420
tccttatttg tggagtctcc aggtcaattg attcagagga tcaccgatga ccctgacgtt
480 agcataaggc tgttagaggt tgtccagagt gaccagtcac aatctggcct
taccttcgca 540 tcaagaggta ccaacatgga ggatgaggcg gaccaatact
tttcacatga tgatccaatt 600 agtagtgatc aatccaggtt cggatggttc
gggaacaagg aaatctcaga tattgaagtg 660 caagaccctg agggattcaa
catgattctg ggtaccatcc tagcccaaat ttgggtcttg 720 ctcgcaaagg
cggttacggc cccagacacg gcagctgatt cggagctaag aaggtggata 780
aagtacaccc aacaaagaag ggtagttggt gaatttagat tggagagaaa atggttggat
840 gtggtgagga acaggattgc cgaggacctc tccttacgcc gattcatggt
cgctctaatc 900 ctggatatca agagaacacc cggaaacaaa cccaggattg
ctgaaatgat atgtgacatt 960 gatacatata tcgtagaggc aggattagcc
agttttatcc tgactattaa gtttgggata 1020 gaaactatgt atcctgctct
tggactgcat gaatttgctg gtgagttatc cacacttgag 1080 tccttgatga
acctttacca gcaaatgggg gaaactgcac cctacatggt aatcctggag 1140
aactcaattc agaacaagtt cagtgcagga tcataccctc tgctctggag ctatgccatg
1200 ggagtaggag tggaacttga aaactccatg ggaggtttga actttggccg
atcttacttt 1260 gatccagcat attttagatt agggcaagag atggtaagga
ggtcagctgg aaaggtcagt 1320 tccacattgg catctgaact cggtatcact
gccgaggatg caaggcttgt ttcagagatt 1380 gcaatgcata ctactgagga
caagatcagt agagcggttg gacccagaca agcccaagta 1440 tcatttctac
acggtgatca aagtgagaat gagctaccga gattgggggg caaggaagat 1500
aggagggtca aacagagtcg aggagaagcc agggagagct acagagaaac cgggcccagc
1560 agagcaagtg atgcgagagc tgcccatctt ccaaccggca cacccctaga
cattgacact 1620 gcaacggagt ccagccaaga tccgcaggac agtcgaaggt
cagctgacgc cctgcttagg 1680 ctgcaagcca tggcaggaat ctcggaagaa
caaggctcag acacggacac ccctatagtg 1740 tacaatgaca gaaatcttct
agactaggtg cgagaggccg agggccagaa caacatccgc 1800 ctaccatcca
tcattgttat aaaaaactta ggaaccaggt ccacacagcc gccagcccat 1860
caaccatcca ctcccacgat tggagccaat ggcagaagag caggcacgcc atgtcaaaaa
1920 cggactggaa tgcatccggg ctctcaaggc cgagcccatc ggctcactgg
ccatcgagga 1980 agctatggca gcatggtcag aaatatcaga caacccagga
caggagcgag ccacctgcag 2040 ggaagagaag gcaggcagtt cgggtctcag
caaaccatgc ctctcagcaa ttggatcaac 2100 tgaaggcggt gcacctcgca
tccgcggtca gggacctgga gagagcgatg acgacgctga 2160 aactttggga
atccccccaa gaaatctcca ggcatcaagc actgggttac agtgttatta 2220
cgtttatgat cacagcggtg aagcggttaa gggaatccaa gatgctgact ctatcatggt
2280 tcaatcaggc cttgatggtg atagcaccct ctcaggagga gacaatgaat
ctgaaaacag 2340 cgatgtggat attggcgaac ctgataccga gggatatgct
atcactgacc ggggatctgc 2400 tcccatctct atggggttca gggcttctga
tgttgaaact gcagaaggag gggagatcca 2460 cgagctcctg agactccaat
ccagaggcaa caactttccg aagcttggga aaactctcaa 2520 tgttcctccg
cccccggacc ccggtagggc cagcacttcc gggacaccca ttaaaaaggg 2580
cacagacgcg agattagcct catttggaac ggagatcgcg tctttattga caggtggtgc
2640 aacccaatgt gctcgaaagt caccctcgga accatcaggg ccaggtgcac
ctgcggggaa 2700 tgtccccgag tgtgtgagca atgccgcact gatacaggag
tggacacccg aatctggtac 2760 cacaatctcc ccgagatccc agaataatga
agaaggggga gactattatg atgatgagct 2820 gttctctgat gtccaagata
ttaaaacagc cttggccaaa atacacgagg ataatcagaa 2880 gataatctcc
aagctagaat cactgctgtt attgaaggga gaagttgagt caattaagaa 2940
gcagatcaac aggcaaaata tcagcatatc caccctggaa ggacacctct caagcatcat
3000 gatcgccatt cctggacttg ggaaggatcc caacgacccc actgcagatg
tcgaaatcaa 3060 tcccgacttg aaacccatca taggcagaga ttcaggccga
gcactggccg aagttctcaa 3120 gaaacccgtt gccagccgac aactccaagg
aatgacaaat ggacggacca gttccagagg 3180 acagctgctg aaggaatttc
agctaaagcc gatcgggaaa aagatgagct cagccgtcgg 3240 gtttgttcct
gacaccggcc ctgcatcacg cagtgtaatc cgctccatta taaaatccag 3300
ccggctagag gaggatcgga agcgttacct gatgactctc cttgatgata tcaaaggagc
3360 caatgatctt gccaagttcc accagatgct gatgaagata ataatgaagt
agctacagct 3420 caacttacct gccaacccca tgccagtcga cccaactagt
acaacctaaa tccattataa 3480 aaaacttagg agcaaagtga ttgcctccca
aggtccacaa tgacagagac ctacgacttc 3540 gacaagtcgg catgggacat
caaagggtcg atcgctccga tacaacccac cacctacagt 3600 gatggcaggc
tggtgcccca ggtcagagtc atagatcctg gtctaggcga caggaaggat 3660
gaatgcttta tgtacatgtt tctgctgggg gttgttgagg acagcgattc cctagggcct
3720 ccaatcgggc gagcatttgg gttcctgccc ttaggtgttg gcagatccac
agcaaagccc 3780 gaaaaactcc tcaaagaggc cactgagctt gacatagttg
ttagacgtac agcagggctc 3840 aatgaaaaac tggtgttcta caacaacacc
ccactaactc tcctcacacc ttggagaaag 3900 gtcctaacaa cagggagtgt
cttcaacgca aaccaagtgt gcaatgcggt taatctgata 3960 ccgctcgata
ccccgcagag gttccgtgtt gtttatatga gcatcacccg tctttcggat 4020
aacgggtatt acaccgttcc tagaagaatg ctggaattca gatcggtcaa tgcagtggcc
4080 ttcaacctgc tggtgaccct taggattgac aaggcgatag gccctgggaa
gatcatcgac 4140 aatacagagc aacttcctga ggcaacattt atggtccaca
tcgggaactt caggagaaag 4200 aagagtgaag tctactctgc cgattattgc
aaaatgaaaa tcgaaaagat gggcctggtt 4260 tttgcacttg gtgggatagg
gggcaccagt cttcacatta gaagcacagg caaaatgagc 4320 aagactctcc
atgcacaact cgggttcaag aagaccttat gttacccgct gatggatatc 4380
aatgaagacc ttaatcgatt actctggagg agcagatgca agatagtaag aatccaggca
4440 gttttgcagc catcagttcc tcaagaattc cgcatttacg acgacgtgat
cataaatgat 4500 gaccaaggac tattcaaagt tctgtagacc gtagtgccca
gcaatgcccg aaaacgaccc 4560 ccctcacaat gacagccaga aggcccggac
aaaaaagccc cctccgaaag actccacgga 4620 ccaagcgaga ggccagccag
cagccgacgg caagcgcgaa caccaggcgg ccccagcaca 4680 gaacagccct
gacacaaggc caccaccagc caccccaatc tgcatcctcc tcgtgggacc 4740
cccgaggacc aacccccaag gctgcccccg atccaaacca ccaaccgcat ccccaccacc
4800 cccgggaaag aaacccccag caattggaag gcccctcccc ctcttcctca
acacaagaac 4860 tccacaaccg aaccgcacaa gcgaccgagg tgacccaacc
gcaggcatcc gactccctag 4920 acagatcctc tctccccggc aaactaaaca
aaacttaggg ccaaggaaca tacacaccca 4980 acagaaccca gaccccggcc
cacggcgccg cgcccccaac ccccgacaac cagagggagc 5040 ccccaaccaa
tcccgccggc tcccccggtg cccacaggca gggacaccaa cccccgaaca 5100
gacccagcac ccaaccatcg acaatccaag acgggggggc ccccccaaaa aaaggccccc
5160 aggggccgac agccagcacc gcgaggaagc ccacccaccc cacacacgac
cacggcaacc 5220 aaaccagaac ccagaccacc ctgggccacc agctcccaga
ctcggccatc accccgcaga 5280 aaggaaaggc cacaacccgc gcaccccagc
cccgatccgg cggggagcca cccaacccga 5340 accagcaccc aagagcgatc
cccgaaggac ccccgaaccg caaaggacat cagtatccca 5400 cagcctctcc
aagtcccccg gtctcctcct cttctcgaag ggaccaaaag atcaatccac 5460
cacacccgac gacactcaac tccccacccc taaaggagac accgggaatc ccagaatcaa
5520 gactcatcca atgtccatca tgggtctcaa ggtgaacgtc tctgccatat
tcatggcagt 5580 actgttaact ctccaaacac ccaccggtca aatccattgg
ggcaatctct ctaagatagg 5640 ggtggtagga ataggaagtg caagctacaa
agttatgact cgttccagcc atcaatcatt 5700 agtcataaaa ttaatgccca
atataactct cctcaataac tgcacgaggg tagagattgc 5760 agaatacagg
agactactga gaacagtttt ggaaccaatt agagatgcac ttaatgcaat 5820
gacccagaat ataagaccgg ttcagagtgt agcttcaagt aggagacaca agagatttgc
5880 gggagtagtc ctggcaggtg cggccctagg cgttgccaca gctgctcaga
taacagccgg 5940 cattgcactt caccagtcca tgctgaactc tcaagccatc
gacaatctga gagcgagcct 6000 ggaaactact aatcaggcaa ttgagacaat
cagacaagca gggcaggaga tgatattggc 6060 tgttcagggt gtccaagact
acatcaataa tgagctgata ccgtctatga accaactatc 6120 ttgtgattta
atcggccaga agctcgggct caaattgctc agatactata cagaaatcct 6180
gtcattattt ggccccagtt tacgggaccc catatctgcg gagatatcta tccaggcttt
6240 gagctatgcg cttggaggag acatcaataa ggtgttagaa aagctcggat
acagtggagg 6300 tgatttactg ggcatcttag agagcggagg aataaaggcc
cggataactc acgtcgacac 6360 agagtcctac ttcattgtcc tcagtatagc
ctatccgacg ctgtccgaga ttaagggggt 6420 gattgtccac cggctagagg
gggtctcgta caacataggc tctcaagagt ggtataccac 6480 tgtgcccaag
tatgttgcaa cccaagggta ccttatctcg aattttgatg agtcatcgtg 6540
tactttcatg ccagagggga ctgtgtgcag ccaaaatgcc ttgtacccga tgagtcctct
6600 gctccaagaa tgcctccggg ggtacaccaa gtcctgtgct cgtacactcg
tatccgggtc 6660 ttttgggaac cggttcattt tatcacaagg gaacctaata
gccaattgtg catcaatcct 6720 ttgcaagtgt tacacaacag gaacgatcat
taatcaagac cctgacaaga tcctaacata 6780 cattgctgcc gatcactgcc
cggtagtcga ggtgaacggc gtgaccatcc aagtcgggag 6840 caggaggtat
ccagacgctg tgtacttgca cagaattgac ctcggtcctc ccatatcatt 6900
ggagaggttg gacgtaggga caaatctggg gaatgcaatt gctaagttgg aggatgccaa
6960 ggaattgttg gagtcatcgg accagatatt gaggagtatg aaaggtttat
cgagcactag 7020 catagtctac atcctgattg cagtgtgtct tggagggttg
atagggatcc ccgctttaat 7080 atgttgctgc agggggcgtt gtaacaaaaa
gggagaacaa gttggtatgt caagaccagg 7140 cctaaagcct gatcttacgg
gaacatcaaa atcctatgta aggtcgctct gatcctctac 7200 aactcttgaa
acacaaatgt cccacaagtc tcctcttcgt catcaagcaa ccaccgcacc 7260
cagcatcaag cccacctgaa attatctccg gcttccctct ggccgaacaa tatcggtagt
7320 taatcaaaac ttagggtgca agatcatcca caatgtcacc acaacgagac
cggataaatg 7380 ccttctacaa agataacccc catcccaagg gaagtaggat
agtcattaac agagaacatc 7440 ttatgattga tagaccttat gttttgctgg
ctgttctgtt tgtcatgttt ctgagcttga 7500 tcgggttgct agccattgca
ggcattagac ttcatcgggc agccatctac accgcagaga 7560 tccataaaag
cctcagcacc aatctagatg taactaactc aatcgagcat caggtcaagg 7620
acgtgctgac accactcttc aaaatcatcg gtgatgaagt gggcctgagg acacctcaga
7680 gattcactga cctagtgaaa ttaatctctg acaagattaa attccttaat
ccggataggg 7740 agtacgactt cagagatctc acttggtgta tcaacccgcc
agagagaatc aaattggatt 7800 atgatcaata ctgtgcagat gtggctgctg
aagagctcat gaatgcattg gtgaactcaa 7860 ctctactgga gaccagaaca
accaatcagt tcctagctgt ctcaaaggga aactgctcag 7920 ggcccactac
aatcagaggt caattctcaa acatgtcgct gtccctgtta gacttgtatt 7980
taggtcgagg ttacaatgtg tcatctatag tcactatgac atcccaggga atgtatgggg
8040 gaacttacct agtggaaaag cctaatctga gcagcaaaag gtcagagttg
tcacaactga 8100 gcatgtaccg agtgtttgaa gtaggtgtta tcagaaatcc
gggtttgggg gctccggtgt 8160 tccatatgac aaactatctt gagcaaccag
tcagtaatga tctcagcaac tgtatggtgg 8220 ctttggggga gctcaaactc
gcagcccttt gtcacgggga agattctatc acaattccct 8280 atcagggatc
agggaaaggt gtcagcttcc agctcgtcaa gctaggtgtc tggaaatccc 8340
caaccgacat gcaatcctgg gtccccttat caacggatga tccagtgata gacaggcttt
8400 acctctcatc tcacagaggt gttatcgctg acaatcaagc aaaatgggct
gtcccgacaa 8460 cacgaacaga tgacaagttg cgaatggaga catgcttcca
acaggcgtgt aagggtaaaa 8520 tccaagcact ctgcgagaat cccgagtggg
caccattgaa ggataacagg attccttcat 8580 acggggtctt gtctgttgat
ctgagtctga cagttgagct taaaatcaaa attgcttcgg 8640 gattcgggcc
attgatcaca cacggttcag ggatggacct atacaaatcc aaccacaaca 8700
atgtgtattg gctgactatc ccgccaatga agaacctagc cttaggtgta atcaacacat
8760 tggagtggat accgagattc aaggttagtc cctacctctt cactgtccca
attaaggaag 8820 caggcgaaga ctgccatgcc ccaacatacc tacctgcgga
ggtggatggt gatgtcaaac 8880 tcagttccaa tctggtgatt ctacctggtc
aagatctcca atatgttttg gcaacctacg 8940 atacttccag ggttgaacat
gctgtggttt attacgttta cagcccaagc cgctcatttt 9000 cttactttta
tccttttagg ttgcctataa agggggtccc catcgaatta caagtggaat 9060
gcttcacatg ggaccaaaaa ctctggtgcc gtcacttctg tgtgcttgcg gactcagaat
9120 ctggtggaca tatcactcac tctgggatgg tgggcatggg agtcagctgc
acagtcaccc 9180 gggaagatgg aaccaatcgc agatagggct gctagtgaac
caatcacatg atgtcaccca 9240 gacatcaggc atacccacta gtgtgaaata
gacatcagaa ttaagaaaaa cgtagggtcc 9300 aagtggttcc ccgttatgga
ctcgctatct gtcaaccaga tcttataccc tgaagttcac 9360 ctagatagcc
cgatagttac caataagata gtagccatcc tggagtatgc tcgagtccct 9420
cacgcttaca gcctggagga ccctacactg tgtcagaaca tcaagcaccg cctaaaaaac
9480 ggattttcca accaaatgat tataaacaat gtggaagttg ggaatgtcat
caagtccaag 9540 cttaggagtt atccggccca ctctcatatt ccatatccaa
attgtaatca ggatttattt 9600 aacatagaag acaaagagtc aacgaggaag
atccgtgaac tcctcaaaaa ggggaattcg 9660 ctgtactcca aagtcagtga
taaggttttc caatgcttaa gggacactaa ctcacggctt 9720 ggcctaggct
ccgaattgag ggaggacatc aaggagaaag ttattaactt gggagtttac 9780
atgcacagct cccagtggtt tgagcccttt ctgttttggt ttacagtcaa gactgagatg
9840 aggtcagtga ttaaatcaca aacccatact tgccatagga ggagacacac
acctgtattc 9900 ttcactggta gttcagttga gttgctaatc tctcgtgacc
ttgttgctat aatcagtaaa 9960 gagtctcaac atgtatatta cctgacattt
gaactggttt tgatgtattg tgatgtcata 10020 gaggggaggt taatgacaga
gaccgctatg actattgatg ctaggtatac agagcttcta 10080 ggaagagtca
gatacatgtg gaaactgata gatggtttct tccctgcact cgggaatcca 10140
acttatcaaa ttgtagccat gctggagcct ctttcacttg cttacctgca gctgagggat
10200 ataacagtag aactcagagg tgctttcctt aaccactgct ttactgaaat
acatgatgtt 10260 cttgaccaaa acgggttttc tgatgaaggt acttatcatg
agttaactga agctctagat 10320 tacattttca taactgatga catacatctg
acaggggaga ttttctcatt tttcagaagt 10380 ttcggccacc ccagacttga
agcagtaacg gctgctgaaa atgttaggaa atacatgaat 10440 cagcctaaag
tcattgtgta tgagactctg atgaaaggtc atgccatatt ttgtggaatc 10500
ataatcaacg gctatcgtga caggcacgga ggcagttggc caccgctgac cctccccctg
10560 catgctgcag acacaatccg gaatgctcaa gcttcaggtg aagggttaac
acatgagcag 10620 tgcgttgata actggaaatc ttttgctgga gtgaaatttg
gctgctttat gcctcttagc 10680 ctggatagtg atctgacaat gtacctaaag
gacaaggcac ttgctgctct ccaaagggaa 10740 tgggattcag tttacccgaa
agagttcctg cgttacgacc ctcccaaggg aaccgggtca 10800 cggaggcttg
tagatgtttt ccttaatgat tcgagctttg acccatatga tgtgataatg 10860
tatgttgtaa gtggagctta cctccatgac cctgagttca acctgtctta cagcctgaaa
10920 gaaaaggaga tcaaggaaac aggtagactt tttgctaaaa tgacttacaa
aatgagggca 10980 tgccaagtga ttgctgaaaa tctaatctca aacgggattg
gcaaatattt taaggacaat 11040 gggatggcca aggatgagca cgatttgact
aaggcactcc acactctagc tgtctcagga 11100 gtccccaaag atctcaaaga
aagtcacagg ggggggccag tcttaaaaac ctactcccga 11160 agcccagtcc
acacaagtac caggaacgtg agagcagcaa aagggtttat agggttccct 11220
caagtaattc ggcaggacca agacactgat catccggaga atatggaagc ttacgagaca
11280 gtcagtgcat ttatcacgac tgatctcaag aagtactgcc ttaattggag
atatgagacc 11340 atcagcttgt ttgcacagag gctaaatgag atttacggat
tgccctcatt tttccagtgg 11400 ctgcataaga ggcttgagac ctctgtcctg
tatgtaagtg accctcattg cccccccgac 11460 cttgacgccc atatcccgtt
atataaagtc cccaatgatc aaatcttcat taagtaccct 11520 atgggaggta
tagaagggta ttgtcagaag ctgtggacca tcagcaccat tccctatcta 11580
tacctggctg cttatgagag cggagtaagg attgcttcgt tagtgcaagg ggacaatcag
11640 accatagccg taacaaaaag ggtacccagc acatggccct acaaccttaa
gaaacgggaa 11700 gctgctagag taactagaga ttactttgta attcttaggc
aaaggctaca tgatattggc 11760 catcacctca aggcaaatga gacaattgtt
tcatcacatt tttttgtcta ttcaaaagga 11820 atatattatg atgggctact
tgtgtcccaa tcactcaaga gcatcgcaag atgtgtattc 11880 tggtcagaga
ctatagttga tgaaacaagg gcagcatgca gtaatattgc tacaacaatg 11940
gctaaaagca tcgagagagg ttatgaccgt taccttgcat attccctgaa cgtcctaaaa
12000 gtgatacagc aaattctgat ctctcttggc ttcacaatca attcaaccat
gacccgggat 12060 gtagtcatac ccctcctcac aaacaacgac ctcttaataa
ggatggcact gttgcccgct 12120 cctattgggg ggatgaatta tctgaatatg
agcaggctgt ttgtcagaaa catcggtgat 12180 ccagtaacat catcaattgc
tgatctcaag agaatgattc tcgcctcact aatgcctgaa 12240 gagaccctcc
atcaagtaat gacacaacaa ccgggggact cttcattcct agactgggct 12300
agcgaccctt actcagcaaa tcttgtatgt gtccagagca tcactagact cctcaagaac
12360 ataactgcaa ggtttgtcct gatccatagt ccaaacccaa tgttaaaagg
attattccat 12420 gatgacagta aagaagagga cgagggactg gcggcattcc
tcatggacag gcatattata 12480 gtacctaggg cagctcatga aatcctggat
catagtgtca caggggcaag agagtctatt 12540 gcaggcatgc tggataccac
aaaaggcttg attcgagcca gcatgaggaa gggggggtta 12600 acctctcgag
tgataaccag attgtccaat tatgactatg aacaattcag agcagggatg 12660
gtgctattga caggaagaaa gagaaatgtc ctcattgaca aagagtcatg ttcagtgcag
12720 ctggcgagag ctctaagaag
ccatatgtgg gcgaggctag ctcgaggacg gcctatttac 12780 ggccttgagg
tccctgatgt actagaatct atgcgaggcc accttattcg gcgtcatgag 12840
acatgtgtca tctgcgagtg tggatcagtc aactacggat ggttttttgt cccctcgggt
12900 tgccaactgg atgatattga caaggaaaca tcatccttga gagtcccata
tattggttct 12960 accactgatg agagaacaga catgaagctt gccttcgtaa
gagccccaag tcgatccttg 13020 cgatctgctg ttagaatagc aacagtgtac
tcatgggctt acggtgatga tgatagctct 13080 tggaacgaag cctggttgtt
ggctaggcaa agggccaatg tgagcctgga ggagctaagg 13140 gtgatcactc
ccatctcaac ttcgactaat ttagcgcata ggttgaggga tcgtagcact 13200
caagtgaaat actcaggtac atcccttgtc cgagtggcga ggtataccac aatctccaac
13260 gacaatctct catttgtcat atcagataag aaggttgata ctaactttat
ataccaacaa 13320 ggaatgcttc tagggttggg tgttttagaa acattgtttc
gactcgagaa agataccgga 13380 tcatctaaca cggtattaca tcttcacgtc
gaaacagatt gttgcgtgat cccgatgata 13440 gatcatccca ggatacccag
ctcccgcaag ctagagctga gggcagagct atgtaccaac 13500 ccattgatat
atgataatgc acctttaatt gacagagatg caacaaggct atacacccag 13560
agccatagga ggcaccttgt ggaatttgtt acatggtcca caccccaact atatcacatt
13620 ttagctaagt ccacagcact atctatgatt gacctggtaa caaaatttga
gaaggaccat 13680 atgaatgaaa tttcagctct cataggggat gacgatatca
atagtttcat aactgagttt 13740 ctgctcatag agccaagatt attcactatc
tacttgggcc agtgtgcggc catcaattgg 13800 gcatttgatg tacattatca
tagaccatca gggaaatatc agatgggtga gctgttgtca 13860 tcgttccttt
ctagaatgag caaaggagtg tttaaggtgc ttgtcaatgc tctaagccac 13920
ccaaagatct acaagaaatt ctggcattgt ggtattatag agcctatcca tggtccttca
13980 cttgatgctc aaaacttgca cacaactgtg tgcaacatgg tttacacatg
ctatatgacc 14040 tacctcgacc tgttgttgaa tgaagagtta gaagagttca
catttctctt gtgtgaaagc 14100 gacgaggatg tagtaccgga cagattcgac
aacatccagg caaaacactt atgtgttctg 14160 gcagatttgt actgtcaacc
agggacctgc ccaccaattc gaggtctaag accggtagag 14220 aaatgtgcag
ttctaaccga ccatatcaag gcagaggcta tgttatctcc agcaggatct 14280
tcgtggaaca taaatccaat tattgtagac cattactcat gctctctgac ttatctccgg
14340 cgaggatcga tcaaacagat aagattgaga gttgatccag gattcatttt
cgacgccctc 14400 gctgaggtaa atgtcagtca gccaaagatc ggcagcaaca
acatctcaaa tatgagcatc 14460 aaggctttca gacccccaca cgatgatgtt
gcaaaattgc tcaaagatat caacacaagc 14520 aagcacaatc ttcccatttc
agggggcaat ctcgccaatt atgaaatcca tgctttccgc 14580 agaatcgggt
tgaactcatc tgcttgctac aaagctgttg agatatcaac attaattagg 14640
agatgccttg agccagggga ggacggcttg ttcttgggtg agggatcggg ttctatgttg
14700 atcacttata aagagatact taaactaaac aagtgcttct ataatagtgg
ggtttccgcc 14760 aattctagat ctggtcaaag ggaattagca ccctatccct
ccgaagttgg ccttgtcgaa 14820 cacagaatgg gagtaggtaa tattgtcaaa
gtgctcttta acgggaggcc cgaagtcacg 14880 tgggtaggca gtgtagattg
cttcaatttc atagttagta atatccctac ctctagtgtg 14940 gggtttatcc
attcagatat agagaccttg cctgacaaag atactataga gaagctagag 15000
gaattggcag ccatcttatc gatggctctg ctcctgggca aaataggatc aatactggtg
15060 attaagctta tgcctttcag cggggatttt gttcagggat ttataagtta
tgtagggtct 15120 cattatagag aagtgaacct tgtataccct agatacagca
acttcatctc tactgaatct 15180 tatttggtta tgacagatct caaggctaac
cggctaatga atcctgaaaa gattaagcag 15240 cagataattg aatcatctgt
gaggacttca cctggactta taggtcacat cctatccatt 15300 aagcaactaa
gctgcataca agcaattgtg ggagacgcag ttagtagagg tgatatcaat 15360
cctactctga aaaaacttac acctatagag caggtgctga tcaattgcgg gttggcaatt
15420 aacggaccta agctgtgcaa agaattgatc caccatgatg ttgcctcagg
gcaagatgga 15480 ttgcttaatt ctatactcat cctctacagg gagttggcaa
gattcaaaga caaccaaaga 15540 agtcaacaag ggatgttcca cgcttacccc
gtattggtaa gtagcaggca acgagaactt 15600 atatctagga tcacccgcaa
attctggggg cacattcttc tttactccgg gaacaaaaag 15660 ttgataaata
agtttatcca gaatctcaag tccggctatc tgatactaga cttacaccag 15720
aatatcttcg ttaagaatct atccaagtca gagaaacaga ttattatgac ggggggtttg
15780 aaacgtgagt gggtttttaa ggtaacagtc aaggagacca aagaatggta
taagttagtc 15840 ggatacagtg ccctgattaa ggactaattg gttgaactcc
ggaaccctaa tcctgcccta 15900 ggtggttagg cattatttgc aatatattaa
agaaaacttt gaaaatacga agtttctatt 15960 cccagctttg tctggtggcc
ggcatggtcc cagcctcctc gctggcgccg gctgggcaac 16020 attccgaggg
gaccgtcccc tcggtaatgg cgaatgggac gcggccgatc cggctgctaa 16080
caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
16140 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
ctatatccgg 16200 atgcggccgc gggccctatg gtacccagct tttgttccct
ttagtgaggg ttaattccga 16260 gcttggcgta atcatggtca tagctgtttc
ctgtgtgaaa ttgttatccg ctcacaattc 16320 cacacaacat aggagccgga
agcataaagt gtaaagcctg gggtgcctaa tgagtgaggt 16380 aactcacatt
aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 16440
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
16500 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
gcggtatcag 16560 ctcactcaaa ggcggtaata cggttatcca cagaatcagg
ggataacgca ggaaagaaca 16620 tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa ggccgcgttg ctggcgtttt 16680 tccataggct cggcccccct
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 16740 gaaacccgac
aggactataa agataccagg cgttcccccc tggaagctcc ctcgtgcgct 16800
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
16860 tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
gttcgctcca 16920 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
ctgcgcctta tccggtaact 16980 atcgtcttga gtccaacccg gtaagacacg
acttatcgcc actggcagca gccactggta 17040 acaggattag cagagcgagg
tatgtaggcg gtgctacaga gttcttgaag tggtggccta 17100 actacggcta
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 17160
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
17220 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
gatcctttga 17280 tcttttctac ggggtctgac gctcagtgga acgaaaactc
acgttaaggg attttggtca 17340 tgagattatc aaaaaggatc ttcacctaga
tccttttaaa ttaaaaatga agttttaaat 17400 caatctaaag tatatatgag
taaacttggt ctgacagtta ccaatgctta atcagtgagg 17460 cacctatctc
agcgatctgt ctatttcgtt catccatagt tgcctgactg cccgtcgtgt 17520
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
17580 acccacgctc accggctcca gatttatcag caataaacca gccagccgga
agggccgagc 17640 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
tattaattgt tgccgggaag 17700 ctagagtaag tagttcgcca gttaatagtt
tgcgcaacgt tgttgccatt gctacaggca 17760 tcgtggtgtc acgctcgtcg
tttggtatgg cttcattcag ctccggttcc caacgatcaa 17820 ggcgagttac
atgatccccc atgttgtgaa aaaaagcggt tagctccttc ggtcctccga 17880
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat gcttatggca gcactgcata
17940 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
tactcaacca 18000 agtcattctg agaatagtgt atgcggcgac cgagttgctc
ttgcccggcg tcaatacggg 18060 ataataccgc gccacatagc agaactttaa
aagtgctcat cattggaaaa cgttcttcgg 18120 ggcgaaaact ctcaaggatc
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 18180 cacccaactg
atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 18240
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
18300 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
agcggataca 18360 tatttgaatg tatttagaaa aataaacaaa taggggttcc
gcgcacattt ccccgaaaag 18420 tgccacctga aattgtaaac gttaatattt
tgttaaaatt cgcgttaaat ttttgttaaa 18480 tcagctcatt ttttaaccaa
taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 18540 agaccgagat
agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 18600
tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
18660 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
cggaacccta 18720 aagggagccc ccgatttaga gcttgacggg gaaagccggc
gaacgtggcg agaaaggaag 18780 ggaagaaagc gaaaggagcg ggcgctaggg
cgctggcaag tgtagcggtc acgctgcgcg 18840 taaccaccac acccgccgcg
cttaatgcgc cgctacaggg cgcgtcccat tcgccattca 18900 ggctgcgcaa
ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccac 18960
cgcggtg 18967 17 1458 DNA Yellow fever virus 17 atgcgagtcg
tgattgccct actggtcttg gctgttggtc cggcctactc agctcactgc 60
attggaatta ctgacaggga tttcattgag ggggtgcatg gaggaacttg ggtttcagct
120 accctggagc aagacaagtg tgtcactgtt atggcccctg acaagccttc
attggacatc 180 tcactagaga cagtagccat tgatagacct gctgaggtga
ggaaagtgtg ttacaatgca 240 gttctcactc atgtgaagat taatgacaag
tgccccagca ctggagaggc ccacctagct 300 gaagagaacg aaggggacaa
tgcgtgcaag cgcacttatt ctgatagagg ctggggcaat 360 ggctgtggcc
tatttgggaa agggagcatt gtggcatgcg ccaaattcac ttgtgccaaa 420
tccatgagtt tgtttgaggt tgatcagacc aaaattcagt atgtcatcag agcacaattg
480 catgtagggg ccaagcagga aaattggact accgacatta agactctcaa
gtttgatgcc 540 ctgtcaggct cccaggaagt cgagttcatt gggtatggaa
aagctacact ggaatgccag 600 gtgcaaactg cggtggactt tggtaacagt
tacatcgctg agatggaaac agagagctgg 660 atagtggaca gacagtgggc
ccaggacttg accctgccat ggcagagtgg aagtggcggg 720 gtgtggagag
agatgcatca tcttgtcgaa tttgaacctc cgcatgccgc cactatcaga 780
gtactggccc tgggaaacca ggaaggctcc ttgaaaacag ctcttactgg cgcaatgagg
840 gttacaaagg acacaaatga caacaacctt tacaaactac atggtggaca
tgtttcttgc 900 agagtgaaat tgtcagcttt gacactcaag gggacatcct
acaaaatatg cactgacaaa 960 atgttttttg tcaagaaccc aactgacact
ggccatggca ctgttgtgat gcaggtgaaa 1020 gtgtcaaaag gagccccctg
caggattcca gtgatagtag ctgatgatct tacagcggca 1080 atcaataaag
gcattttggt tacagttaac cccatcgcct caaccaatga tgatgaagtg 1140
ctgattgagg tgaacccacc ttttggagac agctacatta tcgttgggag aggagattca
1200 cgtctcactt accagtggca caaagaggga agctcaatag gaaagttgtt
cactcagacc 1260 atgaaaggcg tggaacgcct ggccgtcatg ggagacaccg
cctgggattt cagctccgct 1320 ggagggttct tcacttcggt tgggaaagga
attcatacgg tgtttggctc tgcctttcag 1380 gggctatttg gcggcttgaa
ctggataaca aaggtcatca tgggggcggt acttatatgg 1440 gttggcatca
acacataa 1458 18 1488 DNA West Nile virus 18 atgagagttg tgtttgtcgt
gctattgctt ttggtggccc cagcttacag cttcaactgc 60 cttggaatga
gcaacagaga cttcttggaa ggagtgtctg gagcaacatg ggtggatttg 120
gttctcgaag gcgacagctg cgtgactatc atgtctaagg acaagcctac catcgatgtg
180 aagatgatga atatggaggc ggtcaacctg gcagaggtcc gcagttattg
ctatttggct 240 accgtcagcg atctctccac caaagctgcg tgcccgacca
tgggagaagc tcacaatgac 300 aaacgtgctg acccagcttt tgtgtgcaga
caaggagtgg tggacagggg ctggggcaac 360 ggctgcggat tatttggcaa
aggaagcatt gacacatgcg ccaaatttgc ctgctctacc 420 aaggcaatag
gaagaaccat cttgaaagag aatatcaagt acgaagtggc catttttgtc 480
catggaccaa ctactgtgga gtcgcacgga aactactcca cacaggttgg agccactcag
540 gcagggagat tcagcatcac tcctgcggcg ccttcataca cactaaagct
tggagaatat 600 ggagaggtga cagtggactg tgaaccacgg tcagggattg
acaccaatgc atactacgtg 660 atgactgttg gaacaaagac gttcttggtc
catcgtgagt ggttcatgga cctcaacctc 720 ccttggagca gtgctggaag
tactgtgtgg aggaacagag agacgttaat ggagtttgag 780 gaaccacacg
ccacgaagca gtctgtgata gcattgggct cacaagaggg agctctgcat 840
caagctttgg ctggagccat tcctgtggaa ttttcaagca acactgtcaa gttgacgtcg
900 ggtcatttga agtgtagagt gaagatggaa aaattgcagt tgaagggaac
aacctatggc 960 gtctgttcaa aggctttcaa gtttcttggg actcccgcag
acacaggtca cggcactgtg 1020 gtgttggaat tgcagtacac tggcacggat
ggaccttgca aagttcctat ctcgtcagtg 1080 gcttcattga acgacctaac
gccagtgggc agattggtca ctgtcaaccc ttttgtttca 1140 gtggccacgg
ccaacgctaa ggtcctgatt gaattggaac caccctttgg agactcatac 1200
atagtggtgg gcagaggaga acaacagatc aatcaccatt ggcacaagtc tggaagcagc
1260 attggcaaag cctttacaac caccctcaaa ggagcgcaga gactagccgc
tctaggagac 1320 acagcttggg actttggatc agttggaggg gtgttcacct
cagttgggaa ggctgtccat 1380 caagtgttcg gaggagcatt ccgctcactg
ttcggaggca tgtcctggat aacgcaagga 1440 ttgctggggg ctctcctgtt
gtggatgggc atcaatgctc gtgattaa 1488 19 1110 DNA West Nile virus 19
atgaggtcca tagctctcac gtttctcgca gttggaggag ttctgctctt cctctccgtg
60 aacgtgcacg ctgacactgg gtgtgccata gacatcagcc ggcaagagct
gagatgtgga 120 agtggagtgt tcatacacaa tgatgtggag gcttggatgg
accggtacaa gtattaccct 180 gaaacgccac aaggcctagc caagatcatt
cagaaagctc ataaggaagg agtgtgcggt 240 ctacgatcag tttccagact
ggagcatcaa atgtgggaag cagtgaagga cgagctgaac 300 actcttttga
aggagaatgg tgtggacctt agtgtcgtgg ttgagaaaca ggagggaatg 360
tacaagtcag cacctaaacg cctcaccgcc accacggaaa aattggaaat tggctggaag
420 gcctggggaa agagtatttt atttgcacca gaactcgcca acaacacctt
tgtggttgat 480 ggtccggaga ccaaggaatg tccgactcag aatcgcgctt
ggaatagctt agaagtggag 540 gattttggat ttggtctcac cagcactcgg
atgttcctga aggtcagaga gagcaacaca 600 actgaatgtg actcgaagat
cattggaacg gctgtcaaga acaacttggc gatccacagt 660 gacctgtcct
attggattga aagcaggctc aatgatacgt ggaagcttga aagggcagtt 720
ctgggtgaag tcaaatcatg tacgtggcct gagacgcata ccttgtgggg cgatggaatc
780 cttgagagtg acttgataat accagtcaca ctggcgggac cacgaagcaa
tcacaatcgg 840 agacctgggt acaagacaca aaaccagggc ccatgggacg
aaggccgggt agagattgac 900 ttcgattact gcccaggaac tacggtcacc
ctgagtgaga gctgcggaca ccgtggacct 960 gccactcgca ccaccacaga
gagcggaaag ttgataacag attggtgctg caggagctgc 1020 accttaccac
cactgcgcta ccaaactgac agcggctgtt ggtatggtat ggagatcaga 1080
ccacagagac atgatgaaaa gacctaatga 1110 20 33 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 20
tatcgtacga tgagagttgt gtttgtcgtg cta 33 21 30 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 21 atagcgcgct tagacagcct tcccaactga 30 22 1380 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
nucleotide sequence of the WNV env gene 22 atgagagttg tgtttgtcgt
gctattgctt ttggtggccc cagcttacag cttcaactgc 60 cttggaatga
gcaacagaga cttcttggaa ggagtgtctg gagcaacatg ggtggatttg 120
gttctcgaag gcgacagctg cgtgactatc atgtctaagg acaagcctac catcgatgtg
180 aagatgatga atatggaggc ggtcaacctg gcagaggtcc gcagttattg
ctatttggct 240 accgtcagcg atctctccac caaagctgcg tgcccgacca
tgggagaagc tcacaatgac 300 aaacgtgctg acccagcttt tgtgtgcaga
caaggagtgg tggacagggg ctggggcaac 360 ggctgcggat tatttggcaa
aggaagcatt gacacatgcg ccaaatttgc ctgctctacc 420 aaggcaatag
gaagaaccat cttgaaagag aatatcaagt acgaagtggc catttttgtc 480
catggaccaa ctactgtgga gtcgcacgga aactactcca cacaggttgg agccactcag
540 gcagggagat tcagcatcac tcctgcggcg ccttcataca cactaaagct
tggagaatat 600 ggagaggtga cagtggactg tgaaccacgg tcagggattg
acaccaatgc atactacgtg 660 atgactgttg gaacaaagac gttcttggtc
catcgtgagt ggttcatgga cctcaacctc 720 ccttggagca gtgctggaag
tactgtgtgg aggaacagag agacgttaat ggagtttgag 780 gaaccacacg
ccacgaagca gtctgtgata gcattgggct cacaagaggg agctctgcat 840
caagctttgg ctggagccat tcctgtggaa ttttcaagca acactgtcaa gttgacgtcg
900 ggtcatttga agtgtagagt gaagatggaa aaattgcagt tgaagggaac
aacctatggc 960 gtctgttcaa aggctttcaa gtttcttggg actcccgcag
acacaggtca cggcactgtg 1020 gtgttggaat tgcagtacac tggcacggat
ggaccttgca aagttcctat ctcgtcagtg 1080 gcttcattga acgacctaac
gccagtgggc agattggtca ctgtcaaccc ttttgtttca 1140 gtggccacgg
ccaacgctaa ggtcctgatt gaattggaac caccctttgg agactcatac 1200
atagtggtgg gcagaggaga acaacagatc aatcaccatt ggcacaagtc tggaagcagc
1260 attggcaaag cctttacaac caccctcaaa ggagcgcaga gactagccgc
tctaggagac 1320 acagcttggg actttggatc agttggaggg gtgttcacct
cagttgggaa ggctgtctaa 1380 23 459 PRT Artificial Sequence
Description of Artificial Sequence Synthetic WNV polyprotein
sequence 23 Met Arg Val Val Phe Val Val Leu Leu Leu Leu Val Ala Pro
Ala Tyr 1 5 10 15 Ser Phe Asn Cys Leu Gly Met Ser Asn Arg Asp Phe
Leu Glu Gly Val 20 25 30 Ser Gly Ala Thr Trp Val Asp Leu Val Leu
Glu Gly Asp Ser Cys Val 35 40 45 Thr Ile Met Ser Lys Asp Lys Pro
Thr Ile Asp Val Lys Met Met Asn 50 55 60 Met Glu Ala Val Asn Leu
Ala Glu Val Arg Ser Tyr Cys Tyr Leu Ala 65 70 75 80 Thr Val Ser Asp
Leu Ser Thr Lys Ala Ala Cys Pro Thr Met Gly Glu 85 90 95 Ala His
Asn Asp Lys Arg Ala Asp Pro Ala Phe Val Cys Arg Gln Gly 100 105 110
Val Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly 115
120 125 Ser Ile Asp Thr Cys Ala Lys Phe Ala Cys Ser Thr Lys Ala Ile
Gly 130 135 140 Arg Thr Ile Leu Lys Glu Asn Ile Lys Tyr Glu Val Ala
Ile Phe Val 145 150 155 160 His Gly Pro Thr Thr Val Glu Ser His Gly
Asn Tyr Ser Thr Gln Val 165 170 175 Gly Ala Thr Gln Ala Gly Arg Phe
Ser Ile Thr Pro Ala Ala Pro Ser 180 185 190 Tyr Thr Leu Lys Leu Gly
Glu Tyr Gly Glu Val Thr Val Asp Cys Glu 195 200 205 Pro Arg Ser Gly
Ile Asp Thr Asn Ala Tyr Tyr Val Met Thr Val Gly 210 215 220 Thr Lys
Thr Phe Leu Val His Arg Glu Trp Phe Met Asp Leu Asn Leu 225 230 235
240 Pro Trp Ser Ser Ala Gly Ser Thr Val Trp Arg Asn Arg Glu Thr Leu
245 250 255 Met Glu Phe Glu Glu Pro His Ala Thr Lys Gln Ser Val Ile
Ala Leu 260 265 270 Gly Ser Gln Glu Gly Ala Leu His Gln Ala Leu Ala
Gly Ala Ile Pro 275 280 285 Val Glu Phe Ser Ser Asn Thr Val Lys Leu
Thr Ser Gly His Leu Lys 290 295 300 Cys Arg Val Lys Met Glu Lys Leu
Gln Leu Lys Gly Thr Thr Tyr Gly 305 310 315 320 Val Cys Ser Lys Ala
Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly 325 330 335 His Gly Thr
Val Val Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro 340 345 350 Cys
Lys Val Pro Ile Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro 355 360
365 Val Gly Arg Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala
370 375 380 Asn Ala Lys Val Leu Ile Glu Leu Glu Pro Pro Phe Gly Asp
Ser Tyr 385 390 395 400 Ile Val Val Gly Arg Gly Glu Gln Gln Ile Asn
His His Trp His Lys 405 410 415 Ser Gly Ser Ser Ile Gly Lys Ala Phe
Thr Thr Thr Leu Lys Gly Ala 420 425
430 Gln Arg Leu Ala Ala Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val
435 440 445 Gly Gly Val Phe Thr Ser Val Gly Lys Ala Val 450 455 24
2046 DNA Human immunodeficiency virus type 1 24 atgagagtga
aggagaaata tcagcacttg tggagatggg ggtggagatg gggcaccatg 60
ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac agtctattat
120 ggggtacctg tgtggagaga agcaaccacc actctatttt gtgcatcaga
tgctaaagcc 180 tatgatacag aggtacataa tgtttgggcc acacatgcct
gtgtacccac agaccccaac 240 ccacaagaag tagtattggg aaatgtgaca
gaaaatttta acatgtggaa aaataacatg 300 gtagatcaga tgcatgagga
tataatcagt ttatgggatg aaagcctaaa gccatgtgta 360 aaattaaccc
cactctgtgt tactttaaat tgcactaatt tgaatatcac taagaatact 420
actaatctca ctagtagcag ctggggaatg atggaggaag gagaaataaa aaattgctct
480 ttctatatca ccacaagcat aagaaataag gtaaagaaag aatatgcact
ttttaataga 540 cttgatgtag taccagtaaa aaatactagt aatactaagt
ataggttaat aagttgtaac 600 acctcagtca ttacacaggc ctgtccaaag
gtatcctttc agccaattcc catacattat 660 tgtgtcccgg ctgggtttgc
gatactaaag tgtaacaata agacattcaa tggatcagga 720 ccatgcacaa
atgtcagcac agtacaatgt acacatggaa ttaggccagt ggtgtcaact 780
caactgctgt taaatggcag tctagcagaa gaagacatag taattagatc tgaagatttc
840 acagacaatg ttaaaaccat aatagtacag ctaaatgaat ctgtagtaat
taattgtaca 900 agacccaaca acaatacaag agaaaggtta tctataggac
cagggagagc attttatgca 960 agaagaaaca taataggaga tataagacaa
gcacattgta acattagtag agcaaaatgg 1020 aataacactt tacaacagat
agttataaaa ttaagagaaa aatttaggaa taaaacaata 1080 gcctttaatc
aatcctcagg aggggaccca gaaattgtaa tgcacagttt taattgtgga 1140
ggggaatttt tctactgtaa tacagcacaa ctgtttaata gtacttggaa tgttgctgga
1200 gggacaaatg gcactgaagg aaatgacata atcacactcc aatgcagaat
aaaacaaatt 1260 ataaatatgt ggcagaaagt aggaaaagca atgtatgccc
ctcccatcac aggacaaatt 1320 agatgttcat caaatattac agggctgcta
ctaacaagag atggaggtaa tagtactgag 1380 actgagactg agatcttcag
acctggagga ggagatatga gggacaattg gagaagtgaa 1440 ttatataaat
ataaagtagt aagaattgaa ccaataggag tagcacccac cagggcaaag 1500
agaagaacag tgcaaagaga aaaaagagca gtgggaatag gagctgtgtt ccttgggttc
1560 ttgggagcag caggaagcac tatgggcgca gcgtcagtga cgctgacggt
acaggccagg 1620 ctattattgt ctggtatagt gcagcagcag aacaatctgc
tgagggctat tgaggcgcaa 1680 cagaatatgt tgcgactcac agtctggggc
atcaagcagc tccaggcaag agtcctggct 1740 ctggaaagat acctaaggga
tcaacagctc atgggaattt ggggttgctc tggaaaactc 1800 atttgcacca
cttctgtgcc ttggaatgtt agttggagta ataaatctgt ggatgatatt 1860
tggaataaca tgacctggat ggagtgggaa agagaaattg acaattacac agactatata
1920 tatgacttac ttgaaaaatc gcaaacccaa caagaaaaga atgaaaaaga
attattggaa 1980 ttggataaat gggcaagttt gtggaattgg tttgacataa
caaactggct gtggtatata 2040 agataa 2046 25 681 PRT Human
immunodeficiency virus type 1 25 Met Arg Val Lys Glu Lys Tyr Gln
His Leu Trp Arg Trp Gly Trp Arg 1 5 10 15 Trp Gly Thr Met Leu Leu
Gly Met Leu Met Ile Cys Ser Ala Thr Glu 20 25 30 Lys Leu Trp Val
Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Glu Ala 35 40 45 Thr Thr
Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60
Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65
70 75 80 Pro Gln Glu Val Val Leu Gly Asn Val Thr Glu Asn Phe Asn
Met Trp 85 90 95 Lys Asn Asn Met Val Asp Gln Met His Glu Asp Ile
Ile Ser Leu Trp 100 105 110 Asp Glu Ser Leu Lys Pro Cys Val Lys Leu
Thr Pro Leu Cys Val Thr 115 120 125 Leu Asn Cys Thr Asn Leu Asn Ile
Thr Lys Asn Thr Thr Asn Leu Thr 130 135 140 Ser Ser Ser Trp Gly Met
Met Glu Glu Gly Glu Ile Lys Asn Cys Ser 145 150 155 160 Phe Tyr Ile
Thr Thr Ser Ile Arg Asn Lys Val Lys Lys Glu Tyr Ala 165 170 175 Leu
Phe Asn Arg Leu Asp Val Val Pro Val Lys Asn Thr Ser Asn Thr 180 185
190 Lys Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys
195 200 205 Pro Lys Val Ser Phe Gln Pro Ile Pro Ile His Tyr Cys Val
Pro Ala 210 215 220 Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe
Asn Gly Ser Gly 225 230 235 240 Pro Cys Thr Asn Val Ser Thr Val Gln
Cys Thr His Gly Ile Arg Pro 245 250 255 Val Val Ser Thr Gln Leu Leu
Leu Asn Gly Ser Leu Ala Glu Glu Asp 260 265 270 Ile Val Ile Arg Ser
Glu Asp Phe Thr Asp Asn Val Lys Thr Ile Ile 275 280 285 Val Gln Leu
Asn Glu Ser Val Val Ile Asn Cys Thr Arg Pro Asn Asn 290 295 300 Asn
Thr Arg Glu Arg Leu Ser Ile Gly Pro Gly Arg Ala Phe Tyr Ala 305 310
315 320 Arg Arg Asn Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile
Ser 325 330 335 Arg Ala Lys Trp Asn Asn Thr Leu Gln Gln Ile Val Ile
Lys Leu Arg 340 345 350 Glu Lys Phe Arg Asn Lys Thr Ile Ala Phe Asn
Gln Ser Ser Gly Gly 355 360 365 Asp Pro Glu Ile Val Met His Ser Phe
Asn Cys Gly Gly Glu Phe Phe 370 375 380 Tyr Cys Asn Thr Ala Gln Leu
Phe Asn Ser Thr Trp Asn Val Ala Gly 385 390 395 400 Gly Thr Asn Gly
Thr Glu Gly Asn Asp Ile Ile Thr Leu Gln Cys Arg 405 410 415 Ile Lys
Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met Tyr 420 425 430
Ala Pro Pro Ile Thr Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly 435
440 445 Leu Leu Leu Thr Arg Asp Gly Gly Asn Ser Thr Glu Thr Glu Thr
Glu 450 455 460 Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp
Arg Ser Glu 465 470 475 480 Leu Tyr Lys Tyr Lys Val Val Arg Ile Glu
Pro Ile Gly Val Ala Pro 485 490 495 Thr Arg Ala Lys Arg Arg Thr Val
Gln Arg Glu Lys Arg Ala Val Gly 500 505 510 Ile Gly Ala Val Phe Leu
Gly Phe Leu Gly Ala Ala Gly Ser Thr Met 515 520 525 Gly Ala Ala Ser
Val Thr Leu Thr Val Gln Ala Arg Leu Leu Leu Ser 530 535 540 Gly Ile
Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln 545 550 555
560 Gln Asn Met Leu Arg Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala
565 570 575 Arg Val Leu Ala Leu Glu Arg Tyr Leu Arg Asp Gln Gln Leu
Met Gly 580 585 590 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr
Ser Val Pro Trp 595 600 605 Asn Val Ser Trp Ser Asn Lys Ser Val Asp
Asp Ile Trp Asn Asn Met 610 615 620 Thr Trp Met Glu Trp Glu Arg Glu
Ile Asp Asn Tyr Thr Asp Tyr Ile 625 630 635 640 Tyr Asp Leu Leu Glu
Lys Ser Gln Thr Gln Gln Glu Lys Asn Glu Lys 645 650 655 Glu Leu Leu
Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp 660 665 670 Ile
Thr Asn Trp Leu Trp Tyr Ile Arg 675 680 26 2610 DNA Human
immunodeficiency virus type 1 26 atgagagtga aggagaaata tcagcacttg
tggagatggg ggtggagatg gggcaccatg 60 ctccttggga tgttgatgat
ctgtagtgct acagaaaaat tgtgggtcac agtctattat 120 ggggtacctg
tgtggagaga agcaaccacc actctatttt gtgcatcaga tgctaaagcc 180
tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac agaccccaac
240 ccacaagaag tagtattggg aaatgtgaca gaaaatttta acatgtggaa
aaataacatg 300 gtagatcaga tgcatgagga tataatcagt ttatgggatg
aaagcctaaa gccatgtgta 360 aaattaaccc cactctgtgt tactttaaat
tgcactaatt tgaatatcac taagaatact 420 actaatctca ctagtagcag
ctggggaatg atggaggaag gagaaataaa aaattgctct 480 ttctatatca
ccacaagcat aagaaataag gtaaagaaag aatatgcact ttttaataga 540
cttgatgtag taccagtaaa aaatactagt aatactaagt ataggttaat aagttgtaac
600 acctcagtca ttacacaggc ctgtccaaag gtatcctttc agccaattcc
catacattat 660 tgtgtcccgg ctgggtttgc gatactaaag tgtaacaata
agacattcaa tggatcagga 720 ccatgcacaa atgtcagcac agtacaatgt
acacatggaa ttaggccagt ggtgtcaact 780 caactgctgt taaatggcag
tctagcagaa gaagacatag taattagatc tgaagatttc 840 acagacaatg
ttaaaaccat aatagtacag ctaaatgaat ctgtagtaat taattgtaca 900
agacccaaca acaatacaag agaaaggtta tctataggac cagggagagc attttatgca
960 agaagaaaca taataggaga tataagacaa gcacattgta acattagtag
agcaaaatgg 1020 aataacactt tacaacagat agttataaaa ttaagagaaa
aatttaggaa taaaacaata 1080 gcctttaatc aatcctcagg aggggaccca
gaaattgtaa tgcacagttt taattgtgga 1140 ggggaatttt tctactgtaa
tacagcacaa ctgtttaata gtacttggaa tgttgctgga 1200 gggacaaatg
gcactgaagg aaatgacata atcacactcc aatgcagaat aaaacaaatt 1260
ataaatatgt ggcagaaagt aggaaaagca atgtatgccc ctcccatcac aggacaaatt
1320 agatgttcat caaatattac agggctgcta ctaacaagag atggaggtaa
tagtactgag 1380 actgagactg agatcttcag acctggagga ggagatatga
gggacaattg gagaagtgaa 1440 ttatataaat ataaagtagt aagaattgaa
ccaataggag tagcacccac cagggcaaag 1500 agaagaacag tgcaaagaga
aaaaagagca gtgggaatag gagctgtgtt ccttgggttc 1560 ttgggagcag
caggaagcac tatgggcgca gcgtcagtga cgctgacggt acaggccagg 1620
ctattattgt ctggtatagt gcagcagcag aacaatctgc tgagggctat tgaggcgcaa
1680 cagaatatgt tgcgactcac agtctggggc atcaagcagc tccaggcaag
agtcctggct 1740 ctggaaagat acctaaggga tcaacagctc atgggaattt
ggggttgctc tggaaaactc 1800 atttgcacca cttctgtgcc ttggaatgtt
agttggagta ataaatctgt ggatgatatt 1860 tggaataaca tgacctggat
ggagtgggaa agagaaattg acaattacac agactatata 1920 tatgacttac
ttgaaaaatc gcaaacccaa caagaaaaga atgaaaaaga attattggaa 1980
ttggataaat gggcaagttt gtggaattgg tttgacataa caaactggct gtggtatata
2040 agattattca taatgatagt aggaggcttg ataggtttaa gaatagtttt
tgctgtactt 2100 tctatagtaa atagagttag gcagggatat tcaccattat
cgtttcagac cctcctccca 2160 gcctcgaggg gacccgacag gcccgaagga
acagaagaag aaggtggaga gagagacaga 2220 gacagatccg gtccatcagt
gaacggatcc ttggcactta tctgggacga tctgcggagc 2280 ctgtgcctct
tcagctacca ccgcttgaga gacttactct tgattgtaac gaggattgtg 2340
gaacttctgg gacgcagggg gtgggaagcc ctcaaatatt ggtggaatct cctacagtat
2400 tggagtcagg aactaaagaa tagtgctgtt agcttgctac aatatgggtg
gagctatttc 2460 catgaggcgg tccaggccgt ctggagatct gcgacagaga
ctcttgcggg cgcgtgggga 2520 gacttatggg agactcttag gagaggtgga
agatggatac tcgcaatccc caggaggatt 2580 agacaagggc ttgagctcac
tctcttgtga 2610 27 869 PRT Human immunodeficiency virus type 1 27
Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5
10 15 Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr
Glu 20 25 30 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp
Arg Glu Ala 35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys
Ala Tyr Asp Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His Ala
Cys Val Pro Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Gly
Asn Val Thr Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asn Met Val
Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Glu Ser
Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr 115 120 125 Leu
Asn Cys Thr Asn Leu Asn Ile Thr Lys Asn Thr Thr Asn Leu Thr 130 135
140 Ser Ser Ser Trp Gly Met Met Glu Glu Gly Glu Ile Lys Asn Cys Ser
145 150 155 160 Phe Tyr Ile Thr Thr Ser Ile Arg Asn Lys Val Lys Lys
Glu Tyr Ala 165 170 175 Leu Phe Asn Arg Leu Asp Val Val Pro Val Lys
Asn Thr Ser Asn Thr 180 185 190 Lys Tyr Arg Leu Ile Ser Cys Asn Thr
Ser Val Ile Thr Gln Ala Cys 195 200 205 Pro Lys Val Ser Phe Gln Pro
Ile Pro Ile His Tyr Cys Val Pro Ala 210 215 220 Gly Phe Ala Ile Leu
Lys Cys Asn Asn Lys Thr Phe Asn Gly Ser Gly 225 230 235 240 Pro Cys
Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro 245 250 255
Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Asp 260
265 270 Ile Val Ile Arg Ser Glu Asp Phe Thr Asp Asn Val Lys Thr Ile
Ile 275 280 285 Val Gln Leu Asn Glu Ser Val Val Ile Asn Cys Thr Arg
Pro Asn Asn 290 295 300 Asn Thr Arg Glu Arg Leu Ser Ile Gly Pro Gly
Arg Ala Phe Tyr Ala 305 310 315 320 Arg Arg Asn Ile Ile Gly Asp Ile
Arg Gln Ala His Cys Asn Ile Ser 325 330 335 Arg Ala Lys Trp Asn Asn
Thr Leu Gln Gln Ile Val Ile Lys Leu Arg 340 345 350 Glu Lys Phe Arg
Asn Lys Thr Ile Ala Phe Asn Gln Ser Ser Gly Gly 355 360 365 Asp Pro
Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe 370 375 380
Tyr Cys Asn Thr Ala Gln Leu Phe Asn Ser Thr Trp Asn Val Ala Gly 385
390 395 400 Gly Thr Asn Gly Thr Glu Gly Asn Asp Ile Ile Thr Leu Gln
Cys Arg 405 410 415 Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly
Lys Ala Met Tyr 420 425 430 Ala Pro Pro Ile Thr Gly Gln Ile Arg Cys
Ser Ser Asn Ile Thr Gly 435 440 445 Leu Leu Leu Thr Arg Asp Gly Gly
Asn Ser Thr Glu Thr Glu Thr Glu 450 455 460 Ile Phe Arg Pro Gly Gly
Gly Asp Met Arg Asp Asn Trp Arg Ser Glu 465 470 475 480 Leu Tyr Lys
Tyr Lys Val Val Arg Ile Glu Pro Ile Gly Val Ala Pro 485 490 495 Thr
Arg Ala Lys Arg Arg Thr Val Gln Arg Glu Lys Arg Ala Val Gly 500 505
510 Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met
515 520 525 Gly Ala Ala Ser Val Thr Leu Thr Val Gln Ala Arg Leu Leu
Leu Ser 530 535 540 Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala
Ile Glu Ala Gln 545 550 555 560 Gln Asn Met Leu Arg Leu Thr Val Trp
Gly Ile Lys Gln Leu Gln Ala 565 570 575 Arg Val Leu Ala Leu Glu Arg
Tyr Leu Arg Asp Gln Gln Leu Met Gly 580 585 590 Ile Trp Gly Cys Ser
Gly Lys Leu Ile Cys Thr Thr Ser Val Pro Trp 595 600 605 Asn Val Ser
Trp Ser Asn Lys Ser Val Asp Asp Ile Trp Asn Asn Met 610 615 620 Thr
Trp Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr Thr Asp Tyr Ile 625 630
635 640 Tyr Asp Leu Leu Glu Lys Ser Gln Thr Gln Gln Glu Lys Asn Glu
Lys 645 650 655 Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn
Trp Phe Asp 660 665 670 Ile Thr Asn Trp Leu Trp Tyr Ile Arg Leu Phe
Ile Met Ile Val Gly 675 680 685 Gly Leu Ile Gly Leu Arg Ile Val Phe
Ala Val Leu Ser Ile Val Asn 690 695 700 Arg Val Arg Gln Gly Tyr Ser
Pro Leu Ser Phe Gln Thr Leu Leu Pro 705 710 715 720 Ala Ser Arg Gly
Pro Asp Arg Pro Glu Gly Thr Glu Glu Glu Gly Gly 725 730 735 Glu Arg
Asp Arg Asp Arg Ser Gly Pro Ser Val Asn Gly Ser Leu Ala 740 745 750
Leu Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr His Arg 755
760 765 Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu Leu Leu
Gly 770 775 780 Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu
Leu Gln Tyr 785 790 795 800 Trp Ser Gln Glu Leu Lys Asn Ser Ala Val
Ser Leu Leu Gln Tyr Gly 805 810 815 Trp Ser Tyr Phe His Glu Ala Val
Gln Ala Val Trp Arg Ser Ala Thr 820 825 830 Glu Thr Leu Ala Gly Ala
Trp Gly Asp Leu Trp Glu Thr Leu Arg Arg 835 840 845 Gly Gly Arg Trp
Ile Leu Ala Ile Pro Arg Arg Ile Arg Gln Gly Leu 850 855 860 Glu Leu
Thr Leu Leu 865 28 2010 DNA Human immunodeficiency virus type 1 28
atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg gggcaccatg
60 ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac
agtctattat 120 ggggtacctg tgtggagaga agcaaccacc actctatttt
gtgcatcaga tgctaaagcc 180 tatgatacag aggtacataa tgtttgggcc
acacatgcct gtgtacccac agaccccaac 240 ccacaagaag tagtattggg
aaatgtgaca gaaaatttta acatgtggaa aaataacatg 300 gtagatcaga
tgcatgagga tataatcagt ttatgggatg aaagcctaaa gccatgtgta 360
aaattaaccc cactctgtgt tactttaaat tgcactaatt tgaatatcac
taagaatact 420 actaatctca ctagtagcag ctggggaatg atggaggaag
gagaaataaa aaattgctct 480 ttctatatca ccacaagcat aagaaataag
gtaaagaaag aatatgcact ttttaataga 540 cttgatgtag taccagtaaa
aaatactagt aatactaagt ataggttaat aagttgtaac 600 acctcagtca
ttacacaggc ctgtccaaag gtatcctttc agccaattcc catacattat 660
tgtgtcccgg ctgggtttgc gatactaaag tgtaacaata agacattcaa tggatcagga
720 ccatgcacaa atgtcagcac agtacaatgt acacatggaa ttaggccagt
ggtgtcaact 780 caactgctgt taaatggcag tctagcagaa gaagacatag
taattagatc tgaagatttc 840 acagacaatg ttaaaaccat aatagtacag
ctaaatgaat ctgtagtaat taattgtaca 900 agacccaaca acaatgctgc
agaattggat aaatgggcaa gtgctgcaag acaagcacat 960 tgtaacatta
gtagagcaaa atggaataac actttacaac agatagttat aaaattaaga 1020
gaaaaattta ggaataaaac aatagccttt aatcaatcct caggagggga cccagaaatt
1080 gtaatgcaca gttttaattg tggaggggaa tttttctact gtaatacagc
acaactgttt 1140 aatagtactt ggaatgttgc tggagggaca aatggcactg
aaggaaatga cataatcaca 1200 ctccaatgca gaataaaaca aattataaat
atgtggcaga aagtaggaaa agcaatgtat 1260 gcccctccca tcacaggaca
aattagatgt tcatcaaata ttacagggct gctactaaca 1320 agagatggag
gtaatagtac tgagactgag actgagatct tcagacctgg aggaggagat 1380
atgagggaca attggagaag tgaattatat aaatataaag tagtaagaat tgaaccaata
1440 ggagtagcac ccaccagggc aaagagaaga acagtgcaaa gagaaaaaag
agcagtggga 1500 ataggagctg tgttccttgg gttcttggga gcagcaggaa
gcactatggg cgcagcgtca 1560 gtgacgctga cggtacaggc caggctatta
ttgtctggta tagtgcagca gcagaacaat 1620 ctgctgaggg ctattgaggc
gcaacagaat atgttgcgac tcacagtctg gggcatcaag 1680 cagctccagg
caagagtcct ggctctggaa agatacctaa gggatcaaca gctcatggga 1740
atttggggtt gctctggaaa actcatttgc accacttctg tgccttggaa tgttagttgg
1800 agtaataaat ctgtggatga tatttggaat aacatgacct ggatggagtg
ggaaagagaa 1860 attgacaatt acacagacta tatatatgac ttacttgaaa
aatcgcaaac ccaacaagaa 1920 aagaatgaaa aagaattatt ggaattggat
aaatgggcaa gtttgtggaa ttggtttgac 1980 ataacaaact ggctgtggta
tataagataa 2010 29 669 PRT Human immunodeficiency virus type 1 29
Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5
10 15 Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr
Glu 20 25 30 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp
Arg Glu Ala 35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys
Ala Tyr Asp Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His Ala
Cys Val Pro Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Gly
Asn Val Thr Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asn Met Val
Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Glu Ser
Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr 115 120 125 Leu
Asn Cys Thr Asn Leu Asn Ile Thr Lys Asn Thr Thr Asn Leu Thr 130 135
140 Ser Ser Ser Trp Gly Met Met Glu Glu Gly Glu Ile Lys Asn Cys Ser
145 150 155 160 Phe Tyr Ile Thr Thr Ser Ile Arg Asn Lys Val Lys Lys
Glu Tyr Ala 165 170 175 Leu Phe Asn Arg Leu Asp Val Val Pro Val Lys
Asn Thr Ser Asn Thr 180 185 190 Lys Tyr Arg Leu Ile Ser Cys Asn Thr
Ser Val Ile Thr Gln Ala Cys 195 200 205 Pro Lys Val Ser Phe Gln Pro
Ile Pro Ile His Tyr Cys Val Pro Ala 210 215 220 Gly Phe Ala Ile Leu
Lys Cys Asn Asn Lys Thr Phe Asn Gly Ser Gly 225 230 235 240 Pro Cys
Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro 245 250 255
Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Asp 260
265 270 Ile Val Ile Arg Ser Glu Asp Phe Thr Asp Asn Val Lys Thr Ile
Ile 275 280 285 Val Gln Leu Asn Glu Ser Val Val Ile Asn Cys Thr Arg
Pro Asn Asn 290 295 300 Asn Ala Ala Glu Leu Asp Lys Trp Ala Ser Ala
Ala Arg Gln Ala His 305 310 315 320 Cys Asn Ile Ser Arg Ala Lys Trp
Asn Asn Thr Leu Gln Gln Ile Val 325 330 335 Ile Lys Leu Arg Glu Lys
Phe Arg Asn Lys Thr Ile Ala Phe Asn Gln 340 345 350 Ser Ser Gly Gly
Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly 355 360 365 Gly Glu
Phe Phe Tyr Cys Asn Thr Ala Gln Leu Phe Asn Ser Thr Trp 370 375 380
Asn Val Ala Gly Gly Thr Asn Gly Thr Glu Gly Asn Asp Ile Ile Thr 385
390 395 400 Leu Gln Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys
Val Gly 405 410 415 Lys Ala Met Tyr Ala Pro Pro Ile Thr Gly Gln Ile
Arg Cys Ser Ser 420 425 430 Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp
Gly Gly Asn Ser Thr Glu 435 440 445 Thr Glu Thr Glu Ile Phe Arg Pro
Gly Gly Gly Asp Met Arg Asp Asn 450 455 460 Trp Arg Ser Glu Leu Tyr
Lys Tyr Lys Val Val Arg Ile Glu Pro Ile 465 470 475 480 Gly Val Ala
Pro Thr Arg Ala Lys Arg Arg Thr Val Gln Arg Glu Lys 485 490 495 Arg
Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala 500 505
510 Gly Ser Thr Met Gly Ala Ala Ser Val Thr Leu Thr Val Gln Ala Arg
515 520 525 Leu Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu
Arg Ala 530 535 540 Ile Glu Ala Gln Gln Asn Met Leu Arg Leu Thr Val
Trp Gly Ile Lys 545 550 555 560 Gln Leu Gln Ala Arg Val Leu Ala Leu
Glu Arg Tyr Leu Arg Asp Gln 565 570 575 Gln Leu Met Gly Ile Trp Gly
Cys Ser Gly Lys Leu Ile Cys Thr Thr 580 585 590 Ser Val Pro Trp Asn
Val Ser Trp Ser Asn Lys Ser Val Asp Asp Ile 595 600 605 Trp Asn Asn
Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr 610 615 620 Thr
Asp Tyr Ile Tyr Asp Leu Leu Glu Lys Ser Gln Thr Gln Gln Glu 625 630
635 640 Lys Asn Glu Lys Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu
Trp 645 650 655 Asn Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Arg
660 665 30 2574 DNA Human immunodeficiency virus type 1 30
atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg gggcaccatg
60 ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac
agtctattat 120 ggggtacctg tgtggagaga agcaaccacc actctatttt
gtgcatcaga tgctaaagcc 180 tatgatacag aggtacataa tgtttgggcc
acacatgcct gtgtacccac agaccccaac 240 ccacaagaag tagtattggg
aaatgtgaca gaaaatttta acatgtggaa aaataacatg 300 gtagatcaga
tgcatgagga tataatcagt ttatgggatg aaagcctaaa gccatgtgta 360
aaattaaccc cactctgtgt tactttaaat tgcactaatt tgaatatcac taagaatact
420 actaatctca ctagtagcag ctggggaatg atggaggaag gagaaataaa
aaattgctct 480 ttctatatca ccacaagcat aagaaataag gtaaagaaag
aatatgcact ttttaataga 540 cttgatgtag taccagtaaa aaatactagt
aatactaagt ataggttaat aagttgtaac 600 acctcagtca ttacacaggc
ctgtccaaag gtatcctttc agccaattcc catacattat 660 tgtgtcccgg
ctgggtttgc gatactaaag tgtaacaata agacattcaa tggatcagga 720
ccatgcacaa atgtcagcac agtacaatgt acacatggaa ttaggccagt ggtgtcaact
780 caactgctgt taaatggcag tctagcagaa gaagacatag taattagatc
tgaagatttc 840 acagacaatg ttaaaaccat aatagtacag ctaaatgaat
ctgtagtaat taattgtaca 900 agacccaaca acaatgctgc agaattggat
aaatgggcaa gtgctgcaag acaagcacat 960 tgtaacatta gtagagcaaa
atggaataac actttacaac agatagttat aaaattaaga 1020 gaaaaattta
ggaataaaac aatagccttt aatcaatcct caggagggga cccagaaatt 1080
gtaatgcaca gttttaattg tggaggggaa tttttctact gtaatacagc acaactgttt
1140 aatagtactt ggaatgttgc tggagggaca aatggcactg aaggaaatga
cataatcaca 1200 ctccaatgca gaataaaaca aattataaat atgtggcaga
aagtaggaaa agcaatgtat 1260 gcccctccca tcacaggaca aattagatgt
tcatcaaata ttacagggct gctactaaca 1320 agagatggag gtaatagtac
tgagactgag actgagatct tcagacctgg aggaggagat 1380 atgagggaca
attggagaag tgaattatat aaatataaag tagtaagaat tgaaccaata 1440
ggagtagcac ccaccagggc aaagagaaga acagtgcaaa gagaaaaaag agcagtggga
1500 ataggagctg tgttccttgg gttcttggga gcagcaggaa gcactatggg
cgcagcgtca 1560 gtgacgctga cggtacaggc caggctatta ttgtctggta
tagtgcagca gcagaacaat 1620 ctgctgaggg ctattgaggc gcaacagaat
atgttgcgac tcacagtctg gggcatcaag 1680 cagctccagg caagagtcct
ggctctggaa agatacctaa gggatcaaca gctcatggga 1740 atttggggtt
gctctggaaa actcatttgc accacttctg tgccttggaa tgttagttgg 1800
agtaataaat ctgtggatga tatttggaat aacatgacct ggatggagtg ggaaagagaa
1860 attgacaatt acacagacta tatatatgac ttacttgaaa aatcgcaaac
ccaacaagaa 1920 aagaatgaaa aagaattatt ggaattggat aaatgggcaa
gtttgtggaa ttggtttgac 1980 ataacaaact ggctgtggta tataagatta
ttcataatga tagtaggagg cttgataggt 2040 ttaagaatag tttttgctgt
actttctata gtaaatagag ttaggcaggg atattcacca 2100 ttatcgtttc
agaccctcct cccagcctcg aggggacccg acaggcccga aggaacagaa 2160
gaagaaggtg gagagagaga cagagacaga tccggtccat cagtgaacgg atccttggca
2220 cttatctggg acgatctgcg gagcctgtgc ctcttcagct accaccgctt
gagagactta 2280 ctcttgattg taacgaggat tgtggaactt ctgggacgca
gggggtggga agccctcaaa 2340 tattggtgga atctcctaca gtattggagt
caggaactaa agaatagtgc tgttagcttg 2400 ctacaatatg ggtggagcta
tttccatgag gcggtccagg ccgtctggag atctgcgaca 2460 gagactcttg
cgggcgcgtg gggagactta tgggagactc ttaggagagg tggaagatgg 2520
atactcgcaa tccccaggag gattagacaa gggcttgagc tcactctctt gtga 2574 31
857 PRT Human immunodeficiency virus type 1 31 Met Arg Val Lys Glu
Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5 10 15 Trp Gly Thr
Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu 20 25 30 Lys
Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Glu Ala 35 40
45 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu
50 55 60 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp
Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Gly Asn Val Thr Glu Asn
Phe Asn Met Trp 85 90 95 Lys Asn Asn Met Val Asp Gln Met His Glu
Asp Ile Ile Ser Leu Trp 100 105 110 Asp Glu Ser Leu Lys Pro Cys Val
Lys Leu Thr Pro Leu Cys Val Thr 115 120 125 Leu Asn Cys Thr Asn Leu
Asn Ile Thr Lys Asn Thr Thr Asn Leu Thr 130 135 140 Ser Ser Ser Trp
Gly Met Met Glu Glu Gly Glu Ile Lys Asn Cys Ser 145 150 155 160 Phe
Tyr Ile Thr Thr Ser Ile Arg Asn Lys Val Lys Lys Glu Tyr Ala 165 170
175 Leu Phe Asn Arg Leu Asp Val Val Pro Val Lys Asn Thr Ser Asn Thr
180 185 190 Lys Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln
Ala Cys 195 200 205 Pro Lys Val Ser Phe Gln Pro Ile Pro Ile His Tyr
Cys Val Pro Ala 210 215 220 Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys
Thr Phe Asn Gly Ser Gly 225 230 235 240 Pro Cys Thr Asn Val Ser Thr
Val Gln Cys Thr His Gly Ile Arg Pro 245 250 255 Val Val Ser Thr Gln
Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Asp 260 265 270 Ile Val Ile
Arg Ser Glu Asp Phe Thr Asp Asn Val Lys Thr Ile Ile 275 280 285 Val
Gln Leu Asn Glu Ser Val Val Ile Asn Cys Thr Arg Pro Asn Asn 290 295
300 Asn Ala Ala Glu Leu Asp Lys Trp Ala Ser Ala Ala Arg Gln Ala His
305 310 315 320 Cys Asn Ile Ser Arg Ala Lys Trp Asn Asn Thr Leu Gln
Gln Ile Val 325 330 335 Ile Lys Leu Arg Glu Lys Phe Arg Asn Lys Thr
Ile Ala Phe Asn Gln 340 345 350 Ser Ser Gly Gly Asp Pro Glu Ile Val
Met His Ser Phe Asn Cys Gly 355 360 365 Gly Glu Phe Phe Tyr Cys Asn
Thr Ala Gln Leu Phe Asn Ser Thr Trp 370 375 380 Asn Val Ala Gly Gly
Thr Asn Gly Thr Glu Gly Asn Asp Ile Ile Thr 385 390 395 400 Leu Gln
Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly 405 410 415
Lys Ala Met Tyr Ala Pro Pro Ile Thr Gly Gln Ile Arg Cys Ser Ser 420
425 430 Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Ser Thr
Glu 435 440 445 Thr Glu Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met
Arg Asp Asn 450 455 460 Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val
Arg Ile Glu Pro Ile 465 470 475 480 Gly Val Ala Pro Thr Arg Ala Lys
Arg Arg Thr Val Gln Arg Glu Lys 485 490 495 Arg Ala Val Gly Ile Gly
Ala Val Phe Leu Gly Phe Leu Gly Ala Ala 500 505 510 Gly Ser Thr Met
Gly Ala Ala Ser Val Thr Leu Thr Val Gln Ala Arg 515 520 525 Leu Leu
Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala 530 535 540
Ile Glu Ala Gln Gln Asn Met Leu Arg Leu Thr Val Trp Gly Ile Lys 545
550 555 560 Gln Leu Gln Ala Arg Val Leu Ala Leu Glu Arg Tyr Leu Arg
Asp Gln 565 570 575 Gln Leu Met Gly Ile Trp Gly Cys Ser Gly Lys Leu
Ile Cys Thr Thr 580 585 590 Ser Val Pro Trp Asn Val Ser Trp Ser Asn
Lys Ser Val Asp Asp Ile 595 600 605 Trp Asn Asn Met Thr Trp Met Glu
Trp Glu Arg Glu Ile Asp Asn Tyr 610 615 620 Thr Asp Tyr Ile Tyr Asp
Leu Leu Glu Lys Ser Gln Thr Gln Gln Glu 625 630 635 640 Lys Asn Glu
Lys Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp 645 650 655 Asn
Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Arg Leu Phe Ile 660 665
670 Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Val Phe Ala Val Leu
675 680 685 Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser
Phe Gln 690 695 700 Thr Leu Leu Pro Ala Ser Arg Gly Pro Asp Arg Pro
Glu Gly Thr Glu 705 710 715 720 Glu Glu Gly Gly Glu Arg Asp Arg Asp
Arg Ser Gly Pro Ser Val Asn 725 730 735 Gly Ser Leu Ala Leu Ile Trp
Asp Asp Leu Arg Ser Leu Cys Leu Phe 740 745 750 Ser Tyr His Arg Leu
Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val 755 760 765 Glu Leu Leu
Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn 770 775 780 Leu
Leu Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu 785 790
795 800 Leu Gln Tyr Gly Trp Ser Tyr Phe His Glu Ala Val Gln Ala Val
Trp 805 810 815 Arg Ser Ala Thr Glu Thr Leu Ala Gly Ala Trp Gly Asp
Leu Trp Glu 820 825 830 Thr Leu Arg Arg Gly Gly Arg Trp Ile Leu Ala
Ile Pro Arg Arg Ile 835 840 845 Arg Gln Gly Leu Glu Leu Thr Leu Leu
850 855 32 1842 DNA Human immunodeficiency virus type 1 32
atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg gggcaccatg
60 ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac
agtctattat 120 ggggtacctg tgtggagaga agcaaccacc actctatttt
gtgcatcaga tgctaaagcc 180 tatgatacag aggtacataa tgtttgggcc
acacatgcct gtgtacccac agaccccaac 240 ccacaagaag tagtattggg
aaatgtgaca gaaaatttta acatgtggaa aaataacatg 300 gtagatcaga
tgcatgagga tataatcagt ttatgggatg aaagcctaaa gccatgtgta 360
aaattaaccc cactctgtgt tactttaaat tgtaacacct cagtcattac acaggcctgt
420 ccaaaggtat cctttcagcc aattcccata cattattgtg tcccggctgg
gtttgcgata 480 ctaaagtgta acaataagac attcaatgga tcaggaccat
gcacaaatgt cagcacagta 540 caatgtacac atggaattag gccagtggtg
tcaactcaac tgctgttaaa tggcagtcta 600 gcagaagaag acatagtaat
tagatctgaa gatttcacag acaatgttaa aaccataata 660 gtacagctaa
atgaatctgt agtaattaat tgtacaagac ccaacaacaa tacaagagaa 720
aggttatcta taggaccagg gagagcattt tatgcaagaa gaaacataat aggagatata
780 agacaagcac attgtaacat tagtagagca aaatggaata acactttaca
acagatagtt 840 ataaaattaa gagaaaaatt taggaataaa acaatagcct
ttaatcaatc ctcaggaggg 900 gacccagaaa ttgtaatgca cagttttaat
tgtggagggg aatttttcta ctgtaataca 960 gcacaactgt ttaatagtac
ttggaatgtt gctggaggga caaatggcac tgaaggaaat 1020 gacataatca
cactccaatg cagaataaaa caaattataa atatgtggca gaaagtagga 1080
aaagcaatgt atgcccctcc catcacagga caaattagat gttcatcaaa tattacaggg
1140 ctgctactaa caagagatgg aggtaatagt actgagactg agactgagat
cttcagacct 1200 ggaggaggag atatgaggga caattggaga agtgaattat
ataaatataa agtagtaaga 1260 attgaaccaa taggagtagc acccaccagg
gcaaagagaa gaacagtgca
aagagaaaaa 1320 agagcagtgg gaataggagc tgtgttcctt gggttcttgg
gagcagcagg aagcactatg 1380 ggcgcagcgt cagtgacgct gacggtacag
gccaggctat tattgtctgg tatagtgcag 1440 cagcagaaca atctgctgag
ggctattgag gcgcaacaga atatgttgcg actcacagtc 1500 tggggcatca
agcagctcca ggcaagagtc ctggctctgg aaagatacct aagggatcaa 1560
cagctcatgg gaatttgggg ttgctctgga aaactcattt gcaccacttc tgtgccttgg
1620 aatgttagtt ggagtaataa atctgtggat gatatttgga ataacatgac
ctggatggag 1680 tgggaaagag aaattgacaa ttacacagac tatatatatg
acttacttga aaaatcgcaa 1740 acccaacaag aaaagaatga aaaagaatta
ttggaattgg ataaatgggc aagtttgtgg 1800 aattggtttg acataacaaa
ctggctgtgg tatataagat ga 1842 33 613 PRT Human immunodeficiency
virus type 1 33 Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp
Gly Trp Arg 1 5 10 15 Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile
Cys Ser Ala Thr Glu 20 25 30 Lys Leu Trp Val Thr Val Tyr Tyr Gly
Val Pro Val Trp Arg Glu Ala 35 40 45 Thr Thr Thr Leu Phe Cys Ala
Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60 Val His Asn Val Trp
Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu
Val Val Leu Gly Asn Val Thr Glu Asn Phe Asn Met Trp 85 90 95 Lys
Asn Asn Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp 100 105
110 Asp Glu Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr
115 120 125 Leu Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys
Val Ser 130 135 140 Phe Gln Pro Ile Pro Ile His Tyr Cys Val Pro Ala
Gly Phe Ala Ile 145 150 155 160 Leu Lys Cys Asn Asn Lys Thr Phe Asn
Gly Ser Gly Pro Cys Thr Asn 165 170 175 Val Ser Thr Val Gln Cys Thr
His Gly Ile Arg Pro Val Val Ser Thr 180 185 190 Gln Leu Leu Leu Asn
Gly Ser Leu Ala Glu Glu Asp Ile Val Ile Arg 195 200 205 Ser Glu Asp
Phe Thr Asp Asn Val Lys Thr Ile Ile Val Gln Leu Asn 210 215 220 Glu
Ser Val Val Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Glu 225 230
235 240 Arg Leu Ser Ile Gly Pro Gly Arg Ala Phe Tyr Ala Arg Arg Asn
Ile 245 250 255 Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg
Ala Lys Trp 260 265 270 Asn Asn Thr Leu Gln Gln Ile Val Ile Lys Leu
Arg Glu Lys Phe Arg 275 280 285 Asn Lys Thr Ile Ala Phe Asn Gln Ser
Ser Gly Gly Asp Pro Glu Ile 290 295 300 Val Met His Ser Phe Asn Cys
Gly Gly Glu Phe Phe Tyr Cys Asn Thr 305 310 315 320 Ala Gln Leu Phe
Asn Ser Thr Trp Asn Val Ala Gly Gly Thr Asn Gly 325 330 335 Thr Glu
Gly Asn Asp Ile Ile Thr Leu Gln Cys Arg Ile Lys Gln Ile 340 345 350
Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met Tyr Ala Pro Pro Ile 355
360 365 Thr Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu
Thr 370 375 380 Arg Asp Gly Gly Asn Ser Thr Glu Thr Glu Thr Glu Ile
Phe Arg Pro 385 390 395 400 Gly Gly Gly Asp Met Arg Asp Asn Trp Arg
Ser Glu Leu Tyr Lys Tyr 405 410 415 Lys Val Val Arg Ile Glu Pro Ile
Gly Val Ala Pro Thr Arg Ala Lys 420 425 430 Arg Arg Thr Val Gln Arg
Glu Lys Arg Ala Val Gly Ile Gly Ala Val 435 440 445 Phe Leu Gly Phe
Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser 450 455 460 Val Thr
Leu Thr Val Gln Ala Arg Leu Leu Leu Ser Gly Ile Val Gln 465 470 475
480 Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln Asn Met Leu
485 490 495 Arg Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val
Leu Ala 500 505 510 Leu Glu Arg Tyr Leu Arg Asp Gln Gln Leu Met Gly
Ile Trp Gly Cys 515 520 525 Ser Gly Lys Leu Ile Cys Thr Thr Ser Val
Pro Trp Asn Val Ser Trp 530 535 540 Ser Asn Lys Ser Val Asp Asp Ile
Trp Asn Asn Met Thr Trp Met Glu 545 550 555 560 Trp Glu Arg Glu Ile
Asp Asn Tyr Thr Asp Tyr Ile Tyr Asp Leu Leu 565 570 575 Glu Lys Ser
Gln Thr Gln Gln Glu Lys Asn Glu Lys Glu Leu Leu Glu 580 585 590 Leu
Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp 595 600
605 Leu Trp Tyr Ile Arg 610 34 2406 DNA Human immunodeficiency
virus type 1 34 atgagagtga aggagaaata tcagcacttg tggagatggg
ggtggagatg gggcaccatg 60 ctccttggga tgttgatgat ctgtagtgct
acagaaaaat tgtgggtcac agtctattat 120 ggggtacctg tgtggagaga
agcaaccacc actctatttt gtgcatcaga tgctaaagcc 180 tatgatacag
aggtacataa tgtttgggcc acacatgcct gtgtacccac agaccccaac 240
ccacaagaag tagtattggg aaatgtgaca gaaaatttta acatgtggaa aaataacatg
300 gtagatcaga tgcatgagga tataatcagt ttatgggatg aaagcctaaa
gccatgtgta 360 aaattaaccc cactctgtgt tactttaaat tgtaacacct
cagtcattac acaggcctgt 420 ccaaaggtat cctttcagcc aattcccata
cattattgtg tcccggctgg gtttgcgata 480 ctaaagtgta acaataagac
attcaatgga tcaggaccat gcacaaatgt cagcacagta 540 caatgtacac
atggaattag gccagtggtg tcaactcaac tgctgttaaa tggcagtcta 600
gcagaagaag acatagtaat tagatctgaa gatttcacag acaatgttaa aaccataata
660 gtacagctaa atgaatctgt agtaattaat tgtacaagac ccaacaacaa
tacaagagaa 720 aggttatcta taggaccagg gagagcattt tatgcaagaa
gaaacataat aggagatata 780 agacaagcac attgtaacat tagtagagca
aaatggaata acactttaca acagatagtt 840 ataaaattaa gagaaaaatt
taggaataaa acaatagcct ttaatcaatc ctcaggaggg 900 gacccagaaa
ttgtaatgca cagttttaat tgtggagggg aatttttcta ctgtaataca 960
gcacaactgt ttaatagtac ttggaatgtt gctggaggga caaatggcac tgaaggaaat
1020 gacataatca cactccaatg cagaataaaa caaattataa atatgtggca
gaaagtagga 1080 aaagcaatgt atgcccctcc catcacagga caaattagat
gttcatcaaa tattacaggg 1140 ctgctactaa caagagatgg aggtaatagt
actgagactg agactgagat cttcagacct 1200 ggaggaggag atatgaggga
caattggaga agtgaattat ataaatataa agtagtaaga 1260 attgaaccaa
taggagtagc acccaccagg gcaaagagaa gaacagtgca aagagaaaaa 1320
agagcagtgg gaataggagc tgtgttcctt gggttcttgg gagcagcagg aagcactatg
1380 ggcgcagcgt cagtgacgct gacggtacag gccaggctat tattgtctgg
tatagtgcag 1440 cagcagaaca atctgctgag ggctattgag gcgcaacaga
atatgttgcg actcacagtc 1500 tggggcatca agcagctcca ggcaagagtc
ctggctctgg aaagatacct aagggatcaa 1560 cagctcatgg gaatttgggg
ttgctctgga aaactcattt gcaccacttc tgtgccttgg 1620 aatgttagtt
ggagtaataa atctgtggat gatatttgga ataacatgac ctggatggag 1680
tgggaaagag aaattgacaa ttacacagac tatatatatg acttacttga aaaatcgcaa
1740 acccaacaag aaaagaatga aaaagaatta ttggaattgg ataaatgggc
aagtttgtgg 1800 aattggtttg acataacaaa ctggctgtgg tatataagat
tattcataat gatagtagga 1860 ggcttgatag gtttaagaat agtttttgct
gtactttcta tagtaaatag agttaggcag 1920 ggatattcac cattatcgtt
tcagaccctc ctcccagcct cgaggggacc cgacaggccc 1980 gaaggaacag
aagaagaagg tggagagaga gacagagaca gatccggtcc atcagtgaac 2040
ggatccttgg cacttatctg ggacgatctg cggagcctgt gcctcttcag ctaccaccgc
2100 ttgagagact tactcttgat tgtaacgagg attgtggaac ttctgggacg
cagggggtgg 2160 gaagccctca aatattggtg gaatctccta cagtattgga
gtcaggaact aaagaatagt 2220 gctgttagct tgctacaata tgggtggagc
tatttccatg aggcggtcca ggccgtctgg 2280 agatctgcga cagagactct
tgcgggcgcg tggggagact tatgggagac tcttaggaga 2340 ggtggaagat
ggatactcgc aatccccagg aggattagac aagggcttga gctcactctc 2400 ttgtga
2406 35 801 PRT Human immunodeficiency virus type 1 35 Met Arg Val
Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5 10 15 Trp
Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu 20 25
30 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Glu Ala
35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp
Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro
Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Gly Asn Val Thr
Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asn Met Val Asp Gln Met
His Glu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Glu Ser Leu Lys Pro
Cys Val Lys Leu Thr Pro Leu Cys Val Thr 115 120 125 Leu Asn Cys Asn
Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val Ser 130 135 140 Phe Gln
Pro Ile Pro Ile His Tyr Cys Val Pro Ala Gly Phe Ala Ile 145 150 155
160 Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Ser Gly Pro Cys Thr Asn
165 170 175 Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val
Ser Thr 180 185 190 Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Asp
Ile Val Ile Arg 195 200 205 Ser Glu Asp Phe Thr Asp Asn Val Lys Thr
Ile Ile Val Gln Leu Asn 210 215 220 Glu Ser Val Val Ile Asn Cys Thr
Arg Pro Asn Asn Asn Thr Arg Glu 225 230 235 240 Arg Leu Ser Ile Gly
Pro Gly Arg Ala Phe Tyr Ala Arg Arg Asn Ile 245 250 255 Ile Gly Asp
Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys Trp 260 265 270 Asn
Asn Thr Leu Gln Gln Ile Val Ile Lys Leu Arg Glu Lys Phe Arg 275 280
285 Asn Lys Thr Ile Ala Phe Asn Gln Ser Ser Gly Gly Asp Pro Glu Ile
290 295 300 Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys
Asn Thr 305 310 315 320 Ala Gln Leu Phe Asn Ser Thr Trp Asn Val Ala
Gly Gly Thr Asn Gly 325 330 335 Thr Glu Gly Asn Asp Ile Ile Thr Leu
Gln Cys Arg Ile Lys Gln Ile 340 345 350 Ile Asn Met Trp Gln Lys Val
Gly Lys Ala Met Tyr Ala Pro Pro Ile 355 360 365 Thr Gly Gln Ile Arg
Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr 370 375 380 Arg Asp Gly
Gly Asn Ser Thr Glu Thr Glu Thr Glu Ile Phe Arg Pro 385 390 395 400
Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr 405
410 415 Lys Val Val Arg Ile Glu Pro Ile Gly Val Ala Pro Thr Arg Ala
Lys 420 425 430 Arg Arg Thr Val Gln Arg Glu Lys Arg Ala Val Gly Ile
Gly Ala Val 435 440 445 Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr
Met Gly Ala Ala Ser 450 455 460 Val Thr Leu Thr Val Gln Ala Arg Leu
Leu Leu Ser Gly Ile Val Gln 465 470 475 480 Gln Gln Asn Asn Leu Leu
Arg Ala Ile Glu Ala Gln Gln Asn Met Leu 485 490 495 Arg Leu Thr Val
Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala 500 505 510 Leu Glu
Arg Tyr Leu Arg Asp Gln Gln Leu Met Gly Ile Trp Gly Cys 515 520 525
Ser Gly Lys Leu Ile Cys Thr Thr Ser Val Pro Trp Asn Val Ser Trp 530
535 540 Ser Asn Lys Ser Val Asp Asp Ile Trp Asn Asn Met Thr Trp Met
Glu 545 550 555 560 Trp Glu Arg Glu Ile Asp Asn Tyr Thr Asp Tyr Ile
Tyr Asp Leu Leu 565 570 575 Glu Lys Ser Gln Thr Gln Gln Glu Lys Asn
Glu Lys Glu Leu Leu Glu 580 585 590 Leu Asp Lys Trp Ala Ser Leu Trp
Asn Trp Phe Asp Ile Thr Asn Trp 595 600 605 Leu Trp Tyr Ile Arg Leu
Phe Ile Met Ile Val Gly Gly Leu Ile Gly 610 615 620 Leu Arg Ile Val
Phe Ala Val Leu Ser Ile Val Asn Arg Val Arg Gln 625 630 635 640 Gly
Tyr Ser Pro Leu Ser Phe Gln Thr Leu Leu Pro Ala Ser Arg Gly 645 650
655 Pro Asp Arg Pro Glu Gly Thr Glu Glu Glu Gly Gly Glu Arg Asp Arg
660 665 670 Asp Arg Ser Gly Pro Ser Val Asn Gly Ser Leu Ala Leu Ile
Trp Asp 675 680 685 Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr His Arg
Leu Arg Asp Leu 690 695 700 Leu Leu Ile Val Thr Arg Ile Val Glu Leu
Leu Gly Arg Arg Gly Trp 705 710 715 720 Glu Ala Leu Lys Tyr Trp Trp
Asn Leu Leu Gln Tyr Trp Ser Gln Glu 725 730 735 Leu Lys Asn Ser Ala
Val Ser Leu Leu Gln Tyr Gly Trp Ser Tyr Phe 740 745 750 His Glu Ala
Val Gln Ala Val Trp Arg Ser Ala Thr Glu Thr Leu Ala 755 760 765 Gly
Ala Trp Gly Asp Leu Trp Glu Thr Leu Arg Arg Gly Gly Arg Trp 770 775
780 Ile Leu Ala Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Leu Thr Leu
785 790 795 800 Leu 36 1807 DNA Human immunodeficiency virus type 1
36 atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg
gggcaccatg 60 ctccttggga tgttgatgat ctgtagtgct acagaaaaat
tgtgggtcac agtctattat 120 ggggtacctg tgtggagaga agcaaccacc
actctatttt gtgcatcaga tgctaaagcc 180 tatgatacag aggtacataa
tgtttgggcc acacatgcct gtgtacccac agaccccaac 240 ccacaagaag
tagtattggg aaatgtgaca gaaaatttta acatgtggaa aaataacatg 300
gtagatcaga tgcatgagga tataatcagt ttatgggatg aaagcctaaa gccatgtgta
360 aaattaaccc cactctgtgt tactttaaat tgtaacacct cagtcattac
acaggcctgt 420 ccaaaggtat cctttcagcc aattcccata cattattgtg
tcccggctgg gtttgcgata 480 ctaaagtgta acaataagac attcaatgga
tcaggaccat gcacaaatgt cagcacagta 540 caatgtacac atggaattag
gccagtggtg tcaactcaac tgctgttaaa tggcagtcta 600 gcagaagaag
acatagtaat tagatctgaa gatttcacag acaatgttaa aaccataata 660
gtacagctaa atgaatctgt agtaattaat tgtacaagac ccaacaacaa tgctgcagaa
720 ttggataaat gggcaagtgc tgcaagacaa gcacattgta acattagtag
agcaaaatgg 780 aataacactt tacaacagat agttataaaa ttaagagaaa
aatttaggaa taaaacaata 840 gcctttaatc aatcctcagg aggggaccca
gaaattgtaa tgcacagttt taattgtgga 900 ggggaatttt tctactgtaa
tacagcacaa ctgtttaata gtacttggaa tgttgctgga 960 gggacaaatg
gcactgaagg aaatgacata atcacactcc aatgcagaat aaaacaaatt 1020
ataaatatgt ggcagaaagt aggaaaagca atgtatgccc ctcccatcac aggacaaatt
1080 agatgttcat caaatattac agggctgcta ctaacaagag atggaggtaa
tagtactgag 1140 actgagactg agatcttcag acctggagga ggagatatga
gggacaattg gagaagtgaa 1200 ttatataaat ataaagtagt aagaattgaa
ccaataggag tagcacccac cagggcaaag 1260 agaagaacag tgcaaagaga
aaaaagagca gtgggaatag gagctgtgtt ccttgggttc 1320 ttgggagcag
caggaagcac tatgggcgca gcgtcagtga cgctgacggt acaggccagg 1380
ctattattgt ctggtatagt gcagcagcag aacaatctgc tgagggctat tgaggcgcaa
1440 cagaatatgt tgcgactcac agtctggggc atcaagcagc tccaggcaag
agtcctggct 1500 ctggaaagat acctaaggga tcaacagctc atgggaattt
ggggttgctc tggaaaactc 1560 atttgcacca cttctgtgcc ttggaatgtt
agttggagta ataaatctgt ggatgatatt 1620 tggaataaca tgacctggat
ggagtgggaa agagaaattg acaattacac agactatata 1680 tatgacttac
ttgaaaaatc gcaaacccaa caagaaaaga atgaaaaaga attattggaa 1740
ttggataaat gggcaagttt gtggaattgg tttgacataa caaactggct gtggtatata
1800 agataat 1807 37 601 PRT Human immunodeficiency virus type 1 37
Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5
10 15 Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr
Glu 20 25 30 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp
Arg Glu Ala 35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys
Ala Tyr Asp Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His Ala
Cys Val Pro Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Gly
Asn Val Thr Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asn Met Val
Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Glu Ser
Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr 115 120 125 Leu
Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val Ser 130 135
140 Phe Gln Pro Ile Pro Ile His Tyr Cys Val Pro Ala Gly Phe Ala Ile
145 150 155 160 Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Ser Gly Pro
Cys Thr Asn 165 170 175 Val Ser Thr Val Gln Cys Thr His Gly Ile Arg
Pro Val Val Ser Thr 180 185 190 Gln Leu Leu Leu Asn Gly Ser Leu Ala
Glu Glu Asp Ile Val Ile Arg 195 200 205
Ser Glu Asp Phe Thr Asp Asn Val Lys Thr Ile Ile Val Gln Leu Asn 210
215 220 Glu Ser Val Val Ile Asn Cys Thr Arg Pro Asn Asn Asn Ala Ala
Glu 225 230 235 240 Leu Asp Lys Trp Ala Ser Ala Ala Arg Gln Ala His
Cys Asn Ile Ser 245 250 255 Arg Ala Lys Trp Asn Asn Thr Leu Gln Gln
Ile Val Ile Lys Leu Arg 260 265 270 Glu Lys Phe Arg Asn Lys Thr Ile
Ala Phe Asn Gln Ser Ser Gly Gly 275 280 285 Asp Pro Glu Ile Val Met
His Ser Phe Asn Cys Gly Gly Glu Phe Phe 290 295 300 Tyr Cys Asn Thr
Ala Gln Leu Phe Asn Ser Thr Trp Asn Val Ala Gly 305 310 315 320 Gly
Thr Asn Gly Thr Glu Gly Asn Asp Ile Ile Thr Leu Gln Cys Arg 325 330
335 Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met Tyr
340 345 350 Ala Pro Pro Ile Thr Gly Gln Ile Arg Cys Ser Ser Asn Ile
Thr Gly 355 360 365 Leu Leu Leu Thr Arg Asp Gly Gly Asn Ser Thr Glu
Thr Glu Thr Glu 370 375 380 Ile Phe Arg Pro Gly Gly Gly Asp Met Arg
Asp Asn Trp Arg Ser Glu 385 390 395 400 Leu Tyr Lys Tyr Lys Val Val
Arg Ile Glu Pro Ile Gly Val Ala Pro 405 410 415 Thr Arg Ala Lys Arg
Arg Thr Val Gln Arg Glu Lys Arg Ala Val Gly 420 425 430 Ile Gly Ala
Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met 435 440 445 Gly
Ala Ala Ser Val Thr Leu Thr Val Gln Ala Arg Leu Leu Leu Ser 450 455
460 Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln
465 470 475 480 Gln Asn Met Leu Arg Leu Thr Val Trp Gly Ile Lys Gln
Leu Gln Ala 485 490 495 Arg Val Leu Ala Leu Glu Arg Tyr Leu Arg Asp
Gln Gln Leu Met Gly 500 505 510 Ile Trp Gly Cys Ser Gly Lys Leu Ile
Cys Thr Thr Ser Val Pro Trp 515 520 525 Asn Val Ser Trp Ser Asn Lys
Ser Val Asp Asp Ile Trp Asn Asn Met 530 535 540 Thr Trp Met Glu Trp
Glu Arg Glu Ile Asp Asn Tyr Thr Asp Tyr Ile 545 550 555 560 Tyr Asp
Leu Leu Glu Lys Ser Gln Thr Gln Gln Glu Lys Asn Glu Lys 565 570 575
Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp 580
585 590 Ile Thr Asn Trp Leu Trp Tyr Ile Arg 595 600 38 2370 DNA
Human immunodeficiency virus type 1 38 atgagagtga aggagaaata
tcagcacttg tggagatggg ggtggagatg gggcaccatg 60 ctccttggga
tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac agtctattat 120
ggggtacctg tgtggagaga agcaaccacc actctatttt gtgcatcaga tgctaaagcc
180 tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac
agaccccaac 240 ccacaagaag tagtattggg aaatgtgaca gaaaatttta
acatgtggaa aaataacatg 300 gtagatcaga tgcatgagga tataatcagt
ttatgggatg aaagcctaaa gccatgtgta 360 aaattaaccc cactctgtgt
tactttaaat tgtaacacct cagtcattac acaggcctgt 420 ccaaaggtat
cctttcagcc aattcccata cattattgtg tcccggctgg gtttgcgata 480
ctaaagtgta acaataagac attcaatgga tcaggaccat gcacaaatgt cagcacagta
540 caatgtacac atggaattag gccagtggtg tcaactcaac tgctgttaaa
tggcagtcta 600 gcagaagaag acatagtaat tagatctgaa gatttcacag
acaatgttaa aaccataata 660 gtacagctaa atgaatctgt agtaattaat
tgtacaagac ccaacaacaa tgctgcagaa 720 ttggataaat gggcaagtgc
tgcaagacaa gcacattgta acattagtag agcaaaatgg 780 aataacactt
tacaacagat agttataaaa ttaagagaaa aatttaggaa taaaacaata 840
gcctttaatc aatcctcagg aggggaccca gaaattgtaa tgcacagttt taattgtgga
900 ggggaatttt tctactgtaa tacagcacaa ctgtttaata gtacttggaa
tgttgctgga 960 gggacaaatg gcactgaagg aaatgacata atcacactcc
aatgcagaat aaaacaaatt 1020 ataaatatgt ggcagaaagt aggaaaagca
atgtatgccc ctcccatcac aggacaaatt 1080 agatgttcat caaatattac
agggctgcta ctaacaagag atggaggtaa tagtactgag 1140 actgagactg
agatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa 1200
ttatataaat ataaagtagt aagaattgaa ccaataggag tagcacccac cagggcaaag
1260 agaagaacag tgcaaagaga aaaaagagca gtgggaatag gagctgtgtt
ccttgggttc 1320 ttgggagcag caggaagcac tatgggcgca gcgtcagtga
cgctgacggt acaggccagg 1380 ctattattgt ctggtatagt gcagcagcag
aacaatctgc tgagggctat tgaggcgcaa 1440 cagaatatgt tgcgactcac
agtctggggc atcaagcagc tccaggcaag agtcctggct 1500 ctggaaagat
acctaaggga tcaacagctc atgggaattt ggggttgctc tggaaaactc 1560
atttgcacca cttctgtgcc ttggaatgtt agttggagta ataaatctgt ggatgatatt
1620 tggaataaca tgacctggat ggagtgggaa agagaaattg acaattacac
agactatata 1680 tatgacttac ttgaaaaatc gcaaacccaa caagaaaaga
atgaaaaaga attattggaa 1740 ttggataaat gggcaagttt gtggaattgg
tttgacataa caaactggct gtggtatata 1800 agattattca taatgatagt
aggaggcttg ataggtttaa gaatagtttt tgctgtactt 1860 tctatagtaa
atagagttag gcagggatat tcaccattat cgtttcagac cctcctccca 1920
gcctcgaggg gacccgacag gcccgaagga acagaagaag aaggtggaga gagagacaga
1980 gacagatccg gtccatcagt gaacggatcc ttggcactta tctgggacga
tctgcggagc 2040 ctgtgcctct tcagctacca ccgcttgaga gacttactct
tgattgtaac gaggattgtg 2100 gaacttctgg gacgcagggg gtgggaagcc
ctcaaatatt ggtggaatct cctacagtat 2160 tggagtcagg aactaaagaa
tagtgctgtt agcttgctac aatatgggtg gagctatttc 2220 catgaggcgg
tccaggccgt ctggagatct gcgacagaga ctcttgcggg cgcgtgggga 2280
gacttatggg agactcttag gagaggtgga agatggatac tcgcaatccc caggaggatt
2340 agacaagggc ttgagctcac tctcttgtga 2370 39 789 PRT Human
immunodeficiency virus type 1 39 Met Arg Val Lys Glu Lys Tyr Gln
His Leu Trp Arg Trp Gly Trp Arg 1 5 10 15 Trp Gly Thr Met Leu Leu
Gly Met Leu Met Ile Cys Ser Ala Thr Glu 20 25 30 Lys Leu Trp Val
Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Glu Ala 35 40 45 Thr Thr
Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60
Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65
70 75 80 Pro Gln Glu Val Val Leu Gly Asn Val Thr Glu Asn Phe Asn
Met Trp 85 90 95 Lys Asn Asn Met Val Asp Gln Met His Glu Asp Ile
Ile Ser Leu Trp 100 105 110 Asp Glu Ser Leu Lys Pro Cys Val Lys Leu
Thr Pro Leu Cys Val Thr 115 120 125 Leu Asn Cys Asn Thr Ser Val Ile
Thr Gln Ala Cys Pro Lys Val Ser 130 135 140 Phe Gln Pro Ile Pro Ile
His Tyr Cys Val Pro Ala Gly Phe Ala Ile 145 150 155 160 Leu Lys Cys
Asn Asn Lys Thr Phe Asn Gly Ser Gly Pro Cys Thr Asn 165 170 175 Val
Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr 180 185
190 Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Asp Ile Val Ile Arg
195 200 205 Ser Glu Asp Phe Thr Asp Asn Val Lys Thr Ile Ile Val Gln
Leu Asn 210 215 220 Glu Ser Val Val Ile Asn Cys Thr Arg Pro Asn Asn
Asn Ala Ala Glu 225 230 235 240 Leu Asp Lys Trp Ala Ser Ala Ala Arg
Gln Ala His Cys Asn Ile Ser 245 250 255 Arg Ala Lys Trp Asn Asn Thr
Leu Gln Gln Ile Val Ile Lys Leu Arg 260 265 270 Glu Lys Phe Arg Asn
Lys Thr Ile Ala Phe Asn Gln Ser Ser Gly Gly 275 280 285 Asp Pro Glu
Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe 290 295 300 Tyr
Cys Asn Thr Ala Gln Leu Phe Asn Ser Thr Trp Asn Val Ala Gly 305 310
315 320 Gly Thr Asn Gly Thr Glu Gly Asn Asp Ile Ile Thr Leu Gln Cys
Arg 325 330 335 Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys
Ala Met Tyr 340 345 350 Ala Pro Pro Ile Thr Gly Gln Ile Arg Cys Ser
Ser Asn Ile Thr Gly 355 360 365 Leu Leu Leu Thr Arg Asp Gly Gly Asn
Ser Thr Glu Thr Glu Thr Glu 370 375 380 Ile Phe Arg Pro Gly Gly Gly
Asp Met Arg Asp Asn Trp Arg Ser Glu 385 390 395 400 Leu Tyr Lys Tyr
Lys Val Val Arg Ile Glu Pro Ile Gly Val Ala Pro 405 410 415 Thr Arg
Ala Lys Arg Arg Thr Val Gln Arg Glu Lys Arg Ala Val Gly 420 425 430
Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met 435
440 445 Gly Ala Ala Ser Val Thr Leu Thr Val Gln Ala Arg Leu Leu Leu
Ser 450 455 460 Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile
Glu Ala Gln 465 470 475 480 Gln Asn Met Leu Arg Leu Thr Val Trp Gly
Ile Lys Gln Leu Gln Ala 485 490 495 Arg Val Leu Ala Leu Glu Arg Tyr
Leu Arg Asp Gln Gln Leu Met Gly 500 505 510 Ile Trp Gly Cys Ser Gly
Lys Leu Ile Cys Thr Thr Ser Val Pro Trp 515 520 525 Asn Val Ser Trp
Ser Asn Lys Ser Val Asp Asp Ile Trp Asn Asn Met 530 535 540 Thr Trp
Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr Thr Asp Tyr Ile 545 550 555
560 Tyr Asp Leu Leu Glu Lys Ser Gln Thr Gln Gln Glu Lys Asn Glu Lys
565 570 575 Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp
Phe Asp 580 585 590 Ile Thr Asn Trp Leu Trp Tyr Ile Arg Leu Phe Ile
Met Ile Val Gly 595 600 605 Gly Leu Ile Gly Leu Arg Ile Val Phe Ala
Val Leu Ser Ile Val Asn 610 615 620 Arg Val Arg Gln Gly Tyr Ser Pro
Leu Ser Phe Gln Thr Leu Leu Pro 625 630 635 640 Ala Ser Arg Gly Pro
Asp Arg Pro Glu Gly Thr Glu Glu Glu Gly Gly 645 650 655 Glu Arg Asp
Arg Asp Arg Ser Gly Pro Ser Val Asn Gly Ser Leu Ala 660 665 670 Leu
Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr His Arg 675 680
685 Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu Leu Leu Gly
690 695 700 Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu Leu
Gln Tyr 705 710 715 720 Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser
Leu Leu Gln Tyr Gly 725 730 735 Trp Ser Tyr Phe His Glu Ala Val Gln
Ala Val Trp Arg Ser Ala Thr 740 745 750 Glu Thr Leu Ala Gly Ala Trp
Gly Asp Leu Trp Glu Thr Leu Arg Arg 755 760 765 Gly Gly Arg Trp Ile
Leu Ala Ile Pro Arg Arg Ile Arg Gln Gly Leu 770 775 780 Glu Leu Thr
Leu Leu 785 40 1092 DNA Human immunodeficiency virus type 1 40
atgggcgccc gcgccagcgt gctgagcggc ggcgagctgg accgctggga gaagatccgc
60 ctgcgccccg gcggcaagaa gaagtacaag ctgaagcaca tcgtgtgggc
cagccgcgag 120 ctggagcgct tcgccgtgaa ccccggcctg ctggagacca
gcgagggctg ccgccagatc 180 ctgggccagc tgcagcccag cctgcagacc
ggcagcgagg agctgcgcag cctgtacaac 240 accgtggcca ccctgtactg
cgtgcaccag cgcatcgagg tgaaggacac caaggaggcc 300 ctggagaaga
tcgaggagga gcagaacaag agcaagaaga aggcccagca ggccgccgcc 360
gacaccggca acagcagcca agtgagccag aactacccca tcgtgcagaa cctgcagggc
420 cagatggtgc accaggccat cagcccccgc accctgaacg cctgggtgaa
ggtggtggag 480 gagaaggcct tcagccccga ggtgatcccc atgttcagcg
ccctgagcga gggcgccacc 540 ccccaggacc tgaacaccat gctgaacacc
gtgggcggcc accaggccgc catgcagatg 600 ctgaaggaga ccatcaacga
ggaggccgcc gagtgggacc gcctgcaccc cgtgcacgcc 660 ggccccatcg
cccccggcca gatgcgcgag ccccgcggca gcgacatcgc cggcaccacg 720
agcaccctgc aggagcagat cggctggatg accaacaacc cccctatccc cgtgggcgag
780 atctacaagc gctggatcat cctgggcctg aacaagatcg tgcgcatgta
cagccccacg 840 agcatcctgg acatccgcca gggccccaag gagcccttcc
gcgactacgt ggaccgcttc 900 tacaagaccc tgcgggccga gcaggccagc
caggaggtga agaactggat gaccgagacc 960 ctgctggtgc agaacgccaa
ccccgactgc aagaccatcc tgaaggccct gggccccgcc 1020 gccaccctgg
aggagatgat gaccgcctgc cagggcgtgg gcggccccgg ccacaaggcc 1080
cgcgtgctgt aa 1092 41 363 PRT Human immunodeficiency virus type 1
41 Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp
1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys
Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe
Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg
Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser
Glu Glu Leu Arg Ser Leu Tyr Asn 65 70 75 80 Thr Val Ala Thr Leu Tyr
Cys Val His Gln Arg Ile Glu Val Lys Asp 85 90 95 Thr Lys Glu Ala
Leu Glu Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys 100 105 110 Lys Lys
Ala Gln Gln Ala Ala Ala Asp Thr Gly Asn Ser Ser Gln Val 115 120 125
Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His 130
135 140 Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val
Glu 145 150 155 160 Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe
Ser Ala Leu Ser 165 170 175 Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr
Met Leu Asn Thr Val Gly 180 185 190 Gly His Gln Ala Ala Met Gln Met
Leu Lys Glu Thr Ile Asn Glu Glu 195 200 205 Ala Ala Glu Trp Asp Arg
Leu His Pro Val His Ala Gly Pro Ile Ala 210 215 220 Pro Gly Gln Met
Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr 225 230 235 240 Ser
Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile 245 250
255 Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys
260 265 270 Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg
Gln Gly 275 280 285 Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe
Tyr Lys Thr Leu 290 295 300 Arg Ala Glu Gln Ala Ser Gln Glu Val Lys
Asn Trp Met Thr Glu Thr 305 310 315 320 Leu Leu Val Gln Asn Ala Asn
Pro Asp Cys Lys Thr Ile Leu Lys Ala 325 330 335 Leu Gly Pro Ala Ala
Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly 340 345 350 Val Gly Gly
Pro Gly His Lys Ala Arg Val Leu 355 360 42 309 DNA Human
immunodeficiency virus type 1 42 atggagccag tagatcctag actagagccc
tggaagcatc cagggagtaa gcctaaaact 60 gcttgtacca attgctattg
taaaaagtgt tgctttcatt gccaagtttg tttcacaaca 120 aaagccttag
gcatctccta tggcaggaag aagcggagac agcgacgaag agctcatcag 180
aacagtcaga ctcatcaagc ttctctatca aagcagccct cctcccagcc tcgaggggac
240 ccgacaggcc cgaaggaaca gaagaagaag gtggagagag agacagagac
agatccggtc 300 catcagtga 309 43 102 PRT Human immunodeficiency
virus type 1 43 Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His
Pro Gly Ser 1 5 10 15 Lys Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys
Lys Lys Cys Cys Phe 20 25 30 His Cys Gln Val Cys Phe Thr Thr Lys
Ala Leu Gly Ile Ser Tyr Gly 35 40 45 Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala His Gln Asn Ser Gln Thr 50 55 60 His Gln Ala Ser Leu
Ser Lys Gln Pro Ser Ser Gln Pro Arg Gly Asp 65 70 75 80 Pro Thr Gly
Pro Lys Glu Gln Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95 Thr
Asp Pro Val His Gln 100
* * * * *